Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium
ions
P. D. LANGTON, M. T. NELSON, Y. HUANG, AND N. B. STANDEN Department of Physiology, University of Leicester, Leicester LEl 9HN, United Kingdom; and Department of Pharmacology, University of Vermont, Burlington, Vermont 05405
LANGTON, P. D., M. T. NELSON, Y. HUANG, AND N. B. STANDEN.Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium ions. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H927-H934, 1991.The effects of tetraethylammonium ions (TEA+) and tetrapentylammonium ions (TPeA’) on Ca2+-activated K’ (Kc,) channels were studied in membrane patches from mesenteric arterial myocytes. External TEA+ produced a flickery block. The concentration dependence for reduction in mean unitary current was consistent with 1:l binding, with dissociation constants (&) in rat and rabbit of 196 and 159 PM at 0 mV, and the block was weakly voltage dependent. Rate constants for blocking and unblocking were 380 rnM-l= ms-1 and 73 ms-l, respectively. In patches containing several channels TEA+ reduced average current to the same extent as mean unitary current, implying that TEA+ block is independent of the channel state. Block was unaffected by raising external K’ to 120 mM. External TPeA’ blocked with slower kinetics and lower affinity than TEA+ (&, 1.49 mM). The sulfonylurea glibenclamide (lo-100 PM), the hyperpolarizing vasodilator cromakalim (5 PM), and internal ATP (1 mM) were without effect on channel activity. We conclude that TEA+ is a relatively effective blocker of single K ca channels of arterial smooth muscle and should block macroscopic currents equally well, whereas external TPeA+ is about eight times less effective. smooth muscle; tetrapentylammonium; alim
glibenclamide;
cromak-
LARGE-CONDUCTANCECa2+-activated K+ channels (hereafter called Kc, channels) are widespread in smooth muscle and appear to occur at high density in the cell membrane of this tissue (5, 9, 21, 27, 33). Tetraethylammonium ions (TEA+) block many different types of K+ channels with varying degrees of effectiveness (36). External TEA+ is relatively effective in reducing the unitary current amplitude of Kca channels of mammalian muscle and chromaffin cells, with a dissociation constant (KJ in the range 120-300 PM (2, 6, 22, 26, 37, 39), whereas internal TEA+ is much less effective (4, 6, 7, 22). However, although it is widely accepted that TEA+ is an effective blocker of Kca channels in smooth muscle, the stoichiometry, kinetics, and voltage dependence of the blocking reaction of TEA+ are not known. We have therefore characterized the effects of TEA+ on Kca channels of arterial smooth muscle in detail, using patches excised from cells isolated from small branches of rat and rabbit mesenteric arteries. We have investigated the concentration and voltage 0363-6135/91
$1.50
Copyright
dependence and kinetics of block and show that TEA+ block does not lock the channel gate in the open state as happens in ATP-sensitive K+ (KATP) channels of skeletal muscle (15), so that for K ca channels block of macroscopic current should be as effective as that of unitary current. We have also investigated the effects of the larger quaternary ammonium ion tetrapentylammonium (TPeA+) and the sulfonylurea glibenclamide, both of which are very effective at antagonizing the actions of the K+ channel-opening drug cromakalim on vascular smooth muscle. We find that TPeA’ is a less effective blocker of Kc, channels than is TEA+, whereas we could not detect any block by glibenclamide. Cromakalim and internal ATP did not affect channel activity. METHODS Preparation. Rats were killed by stunning followed by decapitation. Rabbits were anesthetized with pentobarbital sodium (35 mg/kg iv) and then exsanguinated. Vascular myocytes were isolated from mesenteric arterial branches 200-300 pm in diameter by enzymatic dissociation. A section of mesenteric artery was removed into cold “isolation buffer” of the following composition (in mM): 140 NaCl, 5 KCl, 1 ethylene glycol-bis@-aminoethyl ether)-N,N,N’,N’-tetracetic acid (EGTA), 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), pH 7.4, adjusted with NaOH. After 30 min the adherent fat was dissected away, and a section with two or three side branches was removed. This tissue was allowed to rest for 15 min in “digest buffer” of the following composition (in mM): 128 NaCl, 5.4 KCl, 4.16 NaHC03, 0.35 Na2HP04, 0.45 KH2PO+ 10 glucose, 2.9 sucrose, 10 HEPES, pH 7.3 with NaOH. The tissue was then tied onto a glass cannula and placed in the barrel of a glass syringe containing 2 ml of the enzyme cocktail. This consisted of collagenase (Sigma Chemical, type IA, 725 U/ml), hyaluronidase (0.3 mg/ml), elastase (Sigma, type IIA, 0.3 mg/ml), and bovine serum albumin (Sigma 7906, 2.5 mg/ml) in digest buffer. The artery was perfused at -0.06 ml/min, and the perfusion solution was collected from the syringe barrel and returned to a reservoir, whence it was recirculated. Both the reservoir and syringe were immersed in a water bath at 35°C. After 20 min, 1 ml of enzyme buffer containing 5 mg trypsin (Sigma, type III) was added, and the tissue was removed 15-20 min later. The dead time from the reservoir to the syringe was 7 min.
0 1991 the American
Physiological
Society
H927
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H928
TEA+
BLOCK
OF
ARTERIAL
SMOOTH
The tissue was removed, and the side branches were cut off. These were gently triturated in isolation buffer until cells could be observed. The solution was then discarded and replaced by three 2-ml changes of digest buffer. The tissue was triturated in the third aliquot until many cells were obtained. Cells were filtered through a 7-mm filter media mat (RS components) to remove tissue debris and plated into 35mm Petri dishes containing 1 ml of digest buffer. Cells were stored at 4°C and used within 24 h (usually within 8 h). Recording methods. Single ionic channels were studied using the inside-out and outside-out configurations of the patch-clamp technique (19). Patch pipettes were pulled from 1.5 mm OD borosilicate capillaries, coated with Sylgard elastomer, and fire polished. Their resistance was in the range 5-20 MQ when filled with electrolyte. Seals, formed by application of negative pressure, were in the order of 10 Gst. Patches of membrane were excised into the standard inside- or outside-out configuration. Single channels were recorded using a List EPC-7 or Axopatch 1C amplifier. Steady holding potentials, and in some cases step command potentials, were applied to the pipette. According to the usual convention, membrane potentials are expressed inside relative to outside, and outward currents are given a positive sign. The solution bathing the cytoplasmic face of inside-out patches or the external face of outside-out patches could be changed by placing them in the flow from a perfusion pipette consisting of a common outlet connected to four different reservoirs (8). Solutions. For experiments, cells were maintained in conventional physiological salt solution containing (in mM) 137 NaCl, 5.4 KCl, 0.44 KHZP04, 0.42 NaH2P04, 4.17 NaHC03, 11.1 glucose, 1.0 MgC12, 1.8 CaC12, 0.05 EGTA, 10 HEPES-NaOH, pH 7.4. Once seals had formed the bath solution was exchanged for low-calcium isolation buffer to reduce the incidence of vesicle formation on excision. For most experiments the patch membrane was exposed to a near physiological K+ gradient, the composition of the two solutions being (in mM) 1) external: 115 NaCl, 5 KCl, 5 EGTA, 10 HEPES, pH 7.4 with NaOH; and 2) internal: 1 K2ATP, 5 EGTA, 10 HEPES-KOH, KC1 to bring [W] to 124 n&l, pH 7.2. The calculated free [Ca”‘] was ~110~~’ M. A variant of this solution contained 4.13 mM CaC12, giving a free [Ca”‘] of 10m6 M. KZATP was added from a fresh, pHadjusted stock solution on the day of the experiment, and reduced the probability of KArP channel openings occurring. For experiments in which symmetrical high K+ solutions were used, both external and internal solutions contained the following (in mM): 91 KCl, 29 KOH, 5 EGTA, 2.93 CaCIZ, 10 HEPES, pH 7.2, giving free [Ca”] of 2 x 10m7 M. TEA Cl or TPeA Cl was added to external solution at concentrations between 50 PM and 2 mM. Glibenclamide was dissolved at 2 mM in ethanol or at 4 mM in a 50:50 mixture of dimethyl sulfoxide (DMSO) and polyethylene glycol (PEG, mol wt 200). These stocks were added to the internal or external solution to give the desired concentration. Cromakalim (BRL 34915) was usually made up as a 2 mM stock in ethanol or at 10 mM in a 50:50 mixture of
MUSCLE
Kca
CHANNELS
DMSO and PEG and then finally into external solution at the desired concentration. The vehicle concentration was usually ~0.5% and did not cause detectable effects on channel activity (n = 4). Data collection and analysis. Single-channel currents were recorded onto FM or video tape with a recording bandwidth of either 20 or 40 kHz. For analysis the tape was replayed through an eight-pole Bessel filter and digitized with a CED 502 or Labmaster DMA TL-40 Ato-D converter and stored on a 286 or 386 PC or a PDP 11/73 computer. The filter cutoff (-3 dB) frequency and digitization rate were normally 3 and 10 kHz, respectively. Mean unitary currents (mJ in the presence of TEA+ were measured by averaging the digitized record, using cursors to select open periods. For control records, amplitudes measured in this way were the same as those measured by fitting Gaussian curves to amplitude histograms. The kinetics of Kca block by external TEA+ were investigated by the method of amplitude distribution analysis (39) as described previously (32). Open times were measured using cursors spaced midway between current levels. In patches containing up to six channels mean open time (&J was calculated by measuring the times (tj) spent at current levels corresponding to j = 1, 2 N channels open, when the maximum likelihood estimate for & is given by (cjN,ltjj)/nC, where n, is the total number of closings (20). To estimate relative channel activity under different conditions, N Popen, where P openis open-state probability, was calculated as ( &E1 tjj)/ T, where T is the duration of the recording. To measure the effect of blocking agents on the mean current in outside-out patches with several Kca channels we measured the mean current directly from the digitized record. Results are expressed as means t SE, and all experiments were performed at room temperature (18-21°C). l
RESULTS
Flickery block by external TEA+. Block by external TEA+ was studied in inside-out patches so that we could change the solution bathing the internal face of the patch to check that the channels were Ca2+ sensitive. TEA+ was included in the pipette solution at 50, 100, 200, or 500 PM. Large-conductance Kca channels were identified by their conductance and voltage and Ca2+ sensitivity. In the absence of TEA+ and in 5 mM extracellular K’/ 124 mM intracellular K+ concentrations, Kca channels had a slope conductance between -10 and +10 mV of 135 pS (Fig. lB), in good agreement with values reported previously in vascular smooth muscle (5). In each patch Ca2+ sensitivity was tested by switching the flow solution bathing the internal face of the membrane to change [Ca”‘] between 10s6 and 10-l’ M (Fig. 1C): only patches in which channel Popen responded rapidly to intracellular Ca2+ concentrations were used. Many patches contained more than one channel, and in these patches we achieved the periods of single-channel activity needed for analysis of the TEA+ block of unitary currents by mixing 10m6 and lo-” M Ca2+ solutions in the flow pipette, adjusting their flow rates to obtain the desired level of activity. Figure 1A shows unitary currents recorded from Kc,
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TEA+
BLOCK
PM TEA
’
-20
Membrane pA
C [Cd’+],
500
”
-40 3.67
-I ms 2 PA
SMOOTH
-*
1 Ow6 M
”
1’
*
4
0
potent%
(n$
d”td &jM _- - - - - -/I. - - - - - -c---
/Al TEA 1.52 C
pA
FIG. 1. External tetraethylammonium ions (TEA+) block of Ca”activated K+ (Kc,) channels. A: recordings from 3 inside-out patches from rat mesenteric arterial myocytes held at 0 mV. Pipette solution contained no TEA+ (top), 100 ,uM TEA+ (middle), 500 PM TEA+ (bottom). C, closed level in each case; broken line, mean amplitude of unitary current; filter, 3 kHz; sample rate, 10 kHz. B: unitary currentvoltage relationship in the absence (a) and presence (A) of 500 FM TEA+. Each point is mean t SE, where this is larger than symbol from 5 patches. Line through control points is drawn by eye and was scaled to fit points in 500 PM TEA+. C: Ca2+ dependence of channel activity in inside-out patch with several channels. Patch was held at 0 mV, and pipette contained 50 ,uM TEA+. Flowing solutions bathing internal face of patch were switched between 10v6 M Ca2’ (solid line) and 10-l’ M Ca” (dashed line). For display, record was filtered at 1 kHz.
channels in a patch in the absence of external TEA+ and from patches in which the pipette solution contained TEA+ at 100 and 500 PM. It can be seen that TEA+ caused a concentration-dependent reduction in the mean single channel current and increased the open channel noise. The rni amplitude at 0 mV was reduced from 5.38 t 0.11 pA (n = 5, rat mesenteric arterial myocytes) in control to 3.67 t 0.13 pA (n = 4) in 100 PM TEA+ and 1.54 t 0.07 pA (n = 5) in 500 PM TEA+. The mean current-voltage relationship in control patches and in patches with 500 FM external TEA+ is shown in Fig. 1B. This type of flickery block is characteristic of a blocking process with relatively rapid kinetics, so that blocking and unblocking events are not fully resolved at the recording frequency used (3 kHz in the case of Fig. 1). Thus a flickery open period in the presence of TEA+ is composed of times spent in both the open and blocked states. The block can be considered in terms of the two general types of blocking model given below CT
k1 k -1
cc,O)’
kb[TEA+]
‘OK
\ l-3
k -b
kb[TEA+]
k
MUSCLE
H929
Kca CHANNELS
(i) will be given by
1.
10
ARTERIAL
B ‘9
Control
100
OF
(scheme 1)
Figure 2A shows the dependence of mJi on TEA+ concentration at 0 mV. The points are well fit by Eq. 1, consistent with 1:l binding of TEA+ to a receptor and with a & (fitted by a least-squares algorithm to results from 18 patches) of 196 PM. The mean of the & values for individual patches from rat myocytes at 0 mV was 193 t 9 PM (n = 18), whereas the value for rabbit myocytes of 159 t 34 PM (n = 9) was not significantly different (P = 0.22, unpaired t test). It can be seen from Fig. 1B that the voltage dependence of the block must be very slight, as the mean current-voltage relationship in 500 PM TEA+ is quite well fit by scaling the control relation. & values were measured at membrane potentials between -40 and +40 mV by fitting m;/i as described above. The dependence of & on membrane potential is shown in Fig. 2B. The line shows a least-squares fit to the Boltzmann equation & = & (0) exp (z 'VF/ RT) with J&(O), the & at 0 mV = 196 ,uM, giving an equivalent valency for the block (x ‘) of 0.06. This suggests that the block by TEA+ shows very slight voltage dependence in the direction expected for an external cation, increasing with hyperpolarization, but that the ion must experience only very little of the membrane voltage field. Kinetics of TEA+ block. Two approaches have been used to extract rate constants from partially resolved blocks of the type seen here. The distribution of current amplitudes in the presence of blocker may be fitted with a ,&function convolved with the Gaussian distribution of the closed level current, varying the block and unblock rates to obtain the best fit (32, 39). Alternatively, the analysis may be done in the frequency domain, by measuring the variance from the difference power spectrum (open minus closed level) of the current noise in the presence of blocker. The ratio of the variance calculated assuming that the blocker causes brief complete channel
A
B 1.0 0.8
\ l
-
0.6 -
i-
0.4 -
1ooJ -40
Membrane
L (C, 0)B
(scheme2)
-b
where C represents closed states of the channel, 0 represents open states, and B represents blocked states. In scheme 1 the blocker binds to the open state and must unbind before the channel can close, whereas in scheme 2 block is independent of the channel state. In each case kb and k-b are the rate constants for blocking and unblocking, so that Kd = k-b/kb. For either scheme, rn; in the presence of TEA+ as a fraction of its control value
-20
0
potential
20
40
(mV)
FIG. 2. Concentration and voltage dependence of TEA’ block in rat arterial myocytes. A: plot of fractional mean unitary current (mJi), at 0 mV, against blocker concentration. Points are means t SE, where larger than symbol from 4-6 patches. Curve is drawn to Eq. 1 which assumes 1:l binding with dissociation constant (J&) of 196 PM. B: voltage dependence of & (e) values from reduction in m;/i (each point is a least-squares fit to results from 14-20 patches, except for that at -40 mV, which is from 3 patches). A, values obtained as rate constants for unblocking and blocking (h-&b) from amplitude distribution analysis (mean t SE of 6-14 values at each voltage). Line is least-squares fit to the Boltzmann function given in text with &(O) = 196 PM and equivalent valency (z ‘) = 0.06.
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H930
TEA+
BLOCK
OF
ARTERIAL
SMOOTH
closings to the measured variance is then used to estimate the corner frequency of the blocking process (29, 32). In the present study, we have concentrated on the amplitude distribution method because this is easier to apply to data in which single channel openings may be quite brief and also because the method gives estimates for both blocking and unblocking rates, allowing calculation of the &, which may then be compared with that measured independently from the reduction in rn; (Fig. ZB). In cases where we were able to use the excess variance method, this gave similar values for the rates. Figure 3 shows current amplitude histograms in a control patch (Fig. 3A) and in a patch exposed to 500 PM TEA+ (Fig. 3B). In the absence of TEA+ both the closed and open current peaks may be well fit with Gaussian distributions. In 500 PM TEA+ the open level peak is moved to a much lower amplitude and the distribution is skewed so that it is no longer Gaussian. Instead, the smooth line fitted to the open level shows a ,& distribution generated using a blocking rate (= hb[TEA’]) of 162 ms-1 and an unblocking rate, h-b, of 57 ms-‘. The blocking rates measured as described above increase with [TEA+] (Fig. 4A), as expected from the models presented in schemes 1 and 2, and the slope of the relationship (which gives the rtzb)is greater at 0 than +40 mV. The k -b did not change significantly with [TEA+], for example, it was 71 t 4 (n = 11, rat myocytes) in 100 PM TEA+ and 65 t 4 (n = 10) in 500 PM TEA+ (P = 0.20, unpaired t test). The dependence of the rate constants kb (calculated as kb/ [TEA’] ) and k-b on voltage (Fig. 4B) shows that the observed small voltage dependence of the Kd appears to reside entirely in the voltage dependence of the blocking reaction. These rate constants were also used to calculate values for & at different voltages. Figure 2B shows that these values agree well with those from measurements of the reduction in Illi.
MUSCLE
Kca CHANNELS
A -
B 250
-iV-I 200 E -
2 ,o
I
fj/y
600 500 400 300
~:::~,~~~,~
0.1
0.3 0.5 [TEA+] (mM)
-20 Membrane
0
20
40
potential
(mV)
FIG. 4. A: dependence of block rate on [TEA’] at 0 mV (0) and +40 mV (A). Points show means (*SE where larger than symbol) of measurements from 11-14 patches. Straight lines are least-squares fits giving blocking rate constants of 399 and 268 mM-‘.rns-’ at 0 mV and +40 mV, respectively. B: rate constants for blocking (kb) (A, units rnM-‘* ms-‘), and unblocking (k-b; l , U/ms-‘) as functions of voltage. Points show means & SE from II-20 patches of rat myocytes.
A Control _.-- I -!ivvq
-.- . , -I- - - -
----- I control
P
= 6.37
pA
C
B ----- ‘TEA+ C
1.0 r
= 3.15
pA
5 pAI f-
10:s -b
\ I-
% F
*
0.4 I 0.2
t
OOL .
A 50
I 100
‘
200
[TEA+1WI 5. TEA’ block in outside-out patches. A: recording from patch held at 0 mV and exposed to control (TEA+-free) external solution. Continuous line shows closed level (c) and broken line mean current level. B: record from same patch in 200 PM external TEA+. C: comparison of reduction in unitary and overall mean current by TEA’. Open bars show m;/i replotted from Fig. 3, whereas solid bars show average current in presence or absence of TEA+ (&rEA+JzontrOJ measured over lo-30 s in outside-out patches like that of A and B above (mean t SE of 4 patches of rat myocytes). FIG.
A 3
B
Control
c
500pM
TEA+
01
0 Current
(PA)
Current
2
4
6
(PA)
FIG. 3. Amplitude distribution analysis of TEA+ block in rat arterial myocytes. A: histogram of current amplitude from patch in absence of external TEA+ held at +20 mV. C and 01, closed and open current levels; curves are Gaussians fitted to these peaks, giving unitary current amplitude of 8.90 pA. B: amplitude histogram from patch with 500 PM external TEA+ and also held at +20 mV. Closed current level is fitted with Gaussian curve, but broadened and skewed open level peak has been fitted by eye with ,&distribution of form f(y) = y’“-I’( 1 - y)(“-‘I/ B(a,b) where B(a,b) = JAy’“-“(l - y)@-l’dy, a = k-b7, b = kb[TEA+]T, and 7 is 0.228/fc, where fc is -3 dB frequency of &pole Bessel filter (1 kHz in this case), convolved with baseline Gaussian (32). Fit gave block rate as 162 ms-’ and unblock rate as 57 ms-‘.
Block of mean currents in outside-out patches by TEA 7 The two types of blocking scheme above have different consequences for the relationship between block of unitary current and the effect of the blocker on macroscopic currents. If block prevents channel closure as in scheme 1, the mean length of an open-blocked burst will increase with blocker concentration, with the total & per burst remaining constant (14,28). The increase in burst length will compensate for the reduction in mean current during the burst, and as a consequence the block of macroscopic current will be slight when Popenis low. The &s for reduction in m; and for block of macroscopic current (& and Ki), respectively, will be related by Ki = KJP,,,,, so block of macroscopic current only becomes as effective as that of unitary current as Popenapproaches unity (15). For the state-independent block of scheme 2, in contrast,
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TEA+
BLOCK
OF
ARTERIAL
SMOOTH
the blocker will reduce unitary and macroscopic currents equally well, irrespective of Popen. External TEA+ block of K+ channels may be of either type: in frog skeletal muscle that of delayed rectifier channels is state independent, whereas TEA+ block of KATP channels presents channel closure (15, 34). We investigated therefore which type of scheme is appropriate for TEA+ block of Kca channels. Patches were excised in the outside-out configuration, using a constant [Ca”‘] of 10B6 M in the internal solution. Such patches normally had several active Kca channels, and we measured the mean current flowing through the patch in different external TEA+ concentrations (Fig. 5). Mean current was measured over periods of lo-30 s. In four patches, channel Popen in control was 0.20,0.41,0.42, and 0.65, so that Ki should have been significantly higher than & for a block of the type in scheme 2. In fact, TEA+ reduced the mean current in outside-out patches by the same amount as it did rni (Fig. 5C). We conclude that the block in this case is of the type shown in scheme 2, and does not prevent channel closure, so that TEA+ should be as effective in blocking macroscopic Kca. currents as it is in blocking single channel currents. TEA+ block in high external K+. Raised external K+ enhances block of Kca channels by external Cs+ (12) but relieves block by internal TEA+ (37). We therefore looked for possible changes in block by external TEA+ when outside-out patches from rat arterial myocytes were bathed in symmetrical 120 mM K+ solutions. Under these conditions, the unitary current-voltage relationship was linear with a slope conductance of 288 pS. Each patch was exposed to TEA+-free solution and to 50, 100, 200, and 500 PM TEA+. Kd values were measured from mi/i and were 164 t 9 ,uM (5 patches, rat myocytes) at -20 mV and 229 t 17 PM (4 patches) at +20 mV. Neither Kd was significantly different from that measured at the
MUSCLE
Kca
CHANNELS
H931
same voltage in 5 mM external K’; these were 172 t 14 PM (14 patches) at -20 mV and 227 t 15 PM (18 patches) at +20 mV (P = 0.73 and 0.97, respectively, unpaired t test). Thus we found no evidence for either enhancement or relief of external TEA+ block by high external K+. Effect of TPeA+. In addition to TEA+, other quaternary ammonium ions block K+ channels, either more or less effectively (17, 36). We investigated the effect of TPeA+ because this has been shown to be a potent blocker of the efflux of 86Rb+ induced by the K+ channel opener cromakalim in rat aorta (31). TPeA+ was applied to the external face of outside-out patches from rat arterial myocytes at concentrations of 0.2, 1.0, and 2.0 mM. Figure 6A shows that TPeA+ blocks with slower kinetics than ‘TEA+, causing a concentration-dependent reduction in open time. The concentration-effect curve for the reduction in mean current by TPeA+ is shown in Fig. 6B and is reasonably well fit assuming 1:l binding with a Kd of 1.49 mM. If an open channel can leave the open state by two routes, closing or blocking, the & will be given by the reciprocal of the sum of the rates for these routes (see, e.g., Ref. 13), so that here & = l/ (kBl + kb[TPeA+]), where k-, is the rate constant for channel closing. Thus l/i0 should be a linear function of [TPeA+]. We did not study the kinetics of TPeA+ block in detail, but the results from two patches, shown in Fig. 6C, suggest that this is so. The slope indicates a kb of 0.36 rnM-‘* ms-‘, and the unblocking rate constant, estimated as kb Kd is 0.54 ms? Effects of glibenclamide, internal A TP, and cromakalim. A widespread feature of the effects of K+ channel opening drugs such as cromakalim is that they are blocked by glibenclamide, a sulfonylurea thought to be specific for KATP channels (1, 16). Glibenclamide is lipid soluble and appears to act when applied to either face of the membrane (1). Because cromakalim has been reported to l
A
cc
Control *
0.8
-
,.! \ I-
0.6
-
0.4
-
0.2
t
.
0.01 0.100
1 .O mM TPeA+
[TP~A+]
e
0.8
; w
0.6
,+o
0.4
\ -
0.2
.
1.000
(mM)
2.0 mM TPeA+
5 PA I 10 ms
0.0”“““““““““““”
FIG. 6. Block by tetrapentylammonium ions (TPeA’). A: records from outside-out patch held at 0 mV and with its external face exposed successively to TPeA+-free solution and to solutions with TPeA’ at 0.2, 1.0, and 2.0 mM. Filter 3 kHz; sample rate, 10 kHz. B: reduction in mean current by TPeA’. Points show average current in presence or absence of TPeA+ ( &TreA+&,ntrol; mean t SE, 4 patches. Line is drawn to Eq. 1 with & = 1.49 mM. C: plot of reciprocal mean open time against [TPeA’]. Symbols show results from 2 different patches of rat myocytes and slope of line gives hb as 0.36 mM-’ ms? l
1 .o
[TP~A+]
2.0
(mM)
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H932
TEA+
BLOCK
OF
ARTERIAL
SMOOTH
MUSCLE
Kca
CHANNELS
A Glibenclamide
(15pM)
1 OOnM Calcium
1
+ *M
I
Cromakalim
III
-
2OpAL
+ 6.wM
Calcium
II
( patch
potential
= +40mV)
Ssec
C
1 mM ATP
B 0.5 lOPAl
0.4
30sec 12
T
I
k 0.3
0
g 0.2 0.1 Current
(PA)
Current
activate Kca channels of smooth muscle (l&23), we have looked for blocking effects of glibenclamide on Kc, channels. In inside-out patches excised from rabbit mesenteric arterial myocytes we could not detect any difference in activity measured as No Popenbetween the absence and presence of glibenclamide at concentrations of lo-20 PM (P = 0.65, paired t test, 8 patches, Fig. 7). In the same patches, glibenclamide did not affect unitary current amplitude, which was 5.44 t 0.34 pA in control and 5.48 + 0.38 pA in glibenclamide (P = 0.64, paired t test). Similarly, in three outside-out patches from rat artery, we detected no effect of external glibenclamide. Mean values were 2.98 t 0.67 in control and 3.01 t 0.66 in 50 PM (2 patches) or 100 PM (1 patch) glibenclamide (P = 0.12, paired t test). Because cromakalim and glibenclamide have been shown to affect K ATp channels in arterial smooth muscle and other tissues (1, 35), we investigated the possibility that there may be functional similarity between KATP and Koa channels by testing the effects of internal ATP (1 mM) on Kca channels (Fig. 7). ATP did not affect the unitary current or Popenof Kc, channels. In six patches N
l
Popen
0
7. A: recording
from inside-out, patch from rabbit mesenteric arterial myocyte held at 0 mV showing effect of 15 FM glibenclamide. B: effects of glibenclamide on 8 inside-out patches as in A above. Activity, measured as N. Popen,was measured over periods of 5-8 min. Open bar shows mean control activity -I- SE and hatched bar shows mean activity in lo-20 PM glibenclamide (3 patches were exposed to 10, 4-15, and l-20 PM). C: lack of effect of internal ATP on Kca channels from rabbit mesenteric artery. Original records with amplitude histograms from longer segments of recordings are shown. N. Popen before and after ATP (as Na2ATP) was 1.80 and 1.77. Internal-free Ca”+ was 6.5 FM and patch potential was 0 mV. The 1 mM ATP was calculated to lower Ca2+ under these conditions from 6.5 to 5.1 PM. Apparently long channel openings displayed in Figs. 7 and 8 are due to program used to plot long segments of record, which leaves out brief closings. FIG.
0
(PA)
+20 +40 +60 Membrane Potential, mV
n O.lpMca 0
gx1 0.1 pM Ca, 5 PM Cromakalim
6.5 PM Ca, 5 FM Cromakalim
FIG. 8. Lack of effect of cromakalim on Kca channels at 4 different voltages. A: recording from inside-out patch from rabbit mesenteric arterial myocyte held at +40 mV, in 0.1 PM Ca2+, in 0.1 PM Ca2+, and 5 PM cromakalim, and in 6.5 PM Ca2+ and 5 PM cromakalim. N-Pop,, of Kca channels in these 3 solutions was 0.005, 0.003, and 2.01, respectively. B: effects of cromakalim (5 FM) on N. Popenat 0, +20, +40, and +60 mV (n = 6 patches). For convenience of display, error bars are not shown at 0 and +20 mV. NoPop,, at 0 mV with and without cromakalim were 0.003 & 0.0008 and 0.002 & 0.0008. N. Popenat +20 mV with and without cromakalim were 0.017 * 0.007 and 0.012 t 0.005. To verify its activity, cromakalim used in each experiment on Kca channels was shown to relax norepinephrine (10 PM)-constricted mesenteric arteries, with ~50 nM cromakalim causing full relaxation.
from rabbit myocytes No Popenwas 1.55 t 0.34 in control and 1.55 t 0.33 in 1 mM internal ATP. We also investigated the effects of cromakalim itself on six patches excised from rabbit arterial myocytes. The cytoplasmic face was exposed to low7 M Ca2+, and cromakalim (5 PM) was also applied to this face. Cromakalim caused a small reduction in channel Popenat all voltages tested (Fig. 8), which was not statistically significant (P > 0.05, paired t test). In three patches from rat myocytes we found no effect of 10 PM cromakalim on channel activity. DISCUSSION
Properties of block by external TEA+. TEA+ produced a flickery block of unitary currents through Kca channels. The & values for the reduction in rni of 196 ,uM in rat and 159 PM in rabbit agree well with those for external TEA+ block of Kca of rat myotubes and chromaffin cells that are in the range 100-300 PM (6, 26, 37, 39). In
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TEA+
BLOCK
OF
ARTERIAL
SMOOTH
smooth muscle & values of 300 PM have been reported in rabbit portal vein (2) and of 100-300 PM in taenia coli (21). We find a 1:l stoichiometry for TEA+ binding, as for block of delayed rectifier and KATP channels by external TEA+ (15, 34). The weak voltage dependence that we find, corresponding to an z’ of 0.06, also seems to be generally true for K+ channel block by external TEA+, with z’ values in the range O-0.2, so that TEA+ seem to enter only a little way into the membrane voltage field to reach its blocking site (6, 15, 34, 37, 39). The & for block by external TEA+ was unaffected by raising external K+ from 5 to 120 mM. Raised external K+ has been reported to enhance the block by external Cs+ of Kea channels reincorporated from rabbit intestinal smooth muscle into lipid bilayers (12). The Cs+ block is strongly voltage dependent, and the enhancement is consistent with a multi-ion pore model for the channel in which K+ can enter behind a blocking Cs+. The weak voltage dependence of block by external TEA+ indicates a much more superficial binding site, preventing a similar effect of external K+. Raised external K+ has also been found to reduce block by internal TEA+ in reincorporated channels from t tubules of skeletal muscle, suggesting competition between K+ and TEA+ for its blocking site (37). The lack of reduction in external TEA+ block by high K+ that we observe exemplifies the difference between the internal and external binding sites for TEA+ (25, 37). Internal block shows greater voltage dependence, and longer chain quaternary ammonium ions are more effective than TEA+ (37). In contrast, the external site is much more specific for TEA+, tetramethylammonium, or tetrapropylammonium for example, being around ~400 less potent (37). We found that external TPeA+ was more effective than these ions, blocking mean current with a & of 1.49 mM, about eightfold higher than that for TEA+. We estimate the rate constants for TEA+ block from ,&function fits to the distributions of current amplitude as 380 t 27 rnM-l ems-’ and 73 t 5 ms-’ for blocking and unblocking, respectively, at 0 mV. TEA+ block of Kca channels of skeletal muscle reincorporated into bilayers has quite similar kinetics, with rate constants of 210 mM-’ l rns-l and 63 ms-’ (37). The kinetics of TEA+ block have also been studied at the single channel level in delayed rectifier and K ATp channels of skeletal muscle. In both these cases TEA+ is much less effective, with & around 7 mM, and the kinetics are faster, so that TEA+ reduces unitary currents without increasing open channel noise (15, 34). TEA+ as blocker of macroscopic Kc, current. Our results show that TEA+ reduction of average current, measured in outside-out patches, follows closely the fractional reduction in unitary current. Because the average current (r) will be given by r = No i. Popen, where i is the unitary current, and Popen is in this case the probability that the channel gate is open, whether or not the channel is blocked by TEA+, the fact that Tand i decline in proportion implies that N* Popen does not change. This suggests that at least under our experimental conditions, external TEA+ blocks the channel without interfering with the channel gating mechanism. Thus external TEA+ should be an effective blocker of macroscopic Kc, current and
MUSCLE
Kca
H933
CHANNELS
because of its quite low Kd may be useful in separating effects on Kca from other K+ currents in smooth muscle. The report that 4 mM TEA+ completely inhibited macroscopic Kca current in rabbit portal vein (3) is consistent with this. In contrast, external TEA+ is less effective at blocking macroscopic currents through KATP channels than it is at reducing unitary current because block prevents channel closure (10, 15). Actions of cromakalim, glibenclamide, and TPeA+ on Kc, channels. Although we could not detect activation of Kca channels of mesenteric artery by the K+ channel opener cromakalim, it has been reported to activate Kca channels in patches excised from vascular smooth muscle and after incorporation into bilayers from rabbit aorta, and it has been suggested that activation of these channels may underlie some of the hyperpolarizing and relaxing action of K+ channel openers on vascular smooth muscle (18, 23). However, these actions are relatively insensitive to TEA+ (2, 35, 38). Because external TEA+ is an effective blocker of Kca channels this argues against their involvement in the functional effects of K+ channel openers. Three other blockers have been found to be potent antagonists of the effects of K+ channel openers; glibenclamide (11, 30, 35, 38); TPeA+, which inhibits cromakalim-induced “Rb+ efflux with a Kd of 0.1 PM (31) and completely reverses cromakalim relaxations of mesenteric arterial rings at 10 PM (24); and Ba2+ (35). We examined the effects of two of these agents on Kc, channels. As discussed above, TPeA+ was less effective than TEA+, whereas we could not detect any effect of glibenclamide at concentrations between 10 and 100 PM on Kca channels. This also argues against the involvement of Kca channels in the effects of K+ channel openers on vascular smooth muscle. We thank Dr. J. Hescheler, who took part in some of the early experiments on rabbit cells, Drs. N. W. Davies and J. M. Quayle for discussions and for comments on the manuscript, and W. King for expert technical assistance. The work was supported by grants from the Wellcome Trust, Medical Research Council, North Atlantic Treaty Organization, American Heart Association (AHA), and National Science Foundation. Y. Huang is supported by a fellowship from AHA, Vermont Affiliate. M. T. Nelson is an Established Investigator of the AHA, and N. B. Standen holds a Wellcome Research Leave Fellowship. Cromakalim was a gift of Beecham Pharmaceuticals. Received
4 June
1990; accepted
in final
form
9 October
1990.
REFERENCES 1. ASHCROFT, F. M. Adenosine 5’-triphosphate-sensitive potassium channels. Annu. Reu. Neurosci. 11: 97-118, 1988. 2. BEECH, D. J., AND T. B. BOLTON. Properties of the cromakaliminduced potassium conductance in smooth muscle cells isolated from the rabbit portal vein. Br. J. Pharmacol. 98: 851-864, 1989. 3. BEECH, D. J., AND T. B. BOLTON. Two components of potassium current activated by depolarization of single smooth muscle cells from the rabbit portal vein. J. Physiol. Lond. 418: 293-309, 1989b. 4. BENHAM,~. D.,T. B. BOLTON, R. J. LANG, AND T. TAKEWAKI. The mechanism of action of Ba2’ and TEA on single Ca’+-activated K’-channels in arterial and intestinal smooth muscle cell membranes. Pfluegers Arch. 403: 120-127, 1985. 5. BENHAM, C. D., T. B. BOLTON, R. J. LANG, AND T. TAKEWAKI. Calcium-activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery. J. Physiol. Lond. 371: 45-67, 1986.
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H934
TEA+
BLOCK
OF
ARTERIAL
SMOOTH
6. BLATZ, A. L., AND K. L. MAGLEBY. Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle. J. Gen. Physiol. 84: l-23, 1984. 7. BLATZ, A. L., AND K. L. MAGLEBY. Calcium-activated potassium channels. Trends Neurosci. 10: 463-467, 1987. 8. CARBONE, E., AND H. D. LUX. Kinetics and selectivity of a lowvoltage-activated calcium current in chick and rat sensory neurones. J. Physiol. Lond. 386: 547-570, 1987. 9. CARL, A., AND K. M. SANDERS. Ca2+-activated K channels of canine colonic myocytes. Am. J. Physiol. 257 (Cell Physiol. 26): C470-C480,1989. 10. CASTLE, N. A., AND D. G. HAYLETT. Effect of channel blockers on potassium efflux from metabolically exhausted frog skeletal muscle. J. Physiol. Lond. 383: 547-570, 1987. 11. CAVERO, I., S. MONDOT, AND M. MESTRE. Vasorelaxant effects of cromakalim in rats are mediated by glibenclamide-sensitive potassium channels. J. PharmacoZ. Exp. Ther. 248: 1261-1268, 1989. 12. CECCHI, X., D. WOLFF, 0. ALVAREZ, AND R. LATORRE. Mechanisms of Cs’ blockade in a Ca2+-activated K channel from smooth muscle. Biophys. J. 52: 707-716, 1987. 13. COLQUHOUN, D., AND A. G. HAWKES. Relaxation and fluctuations of membrane currents that flow through drug-operated channels. Proc. R. Sot. Lond. B Biol. Sci. 199: 231-262, 1977. 14. COLQUHOUN, D., AND A. G. HAWKES. On the stochastic properties of bursts of single ion channel openings and of clusters of bursts. Philos. Trans. R. Sot. Lond. B Biol. Sci. 300: l-59, 1982. 15. DAVIES, N. W., A. E. SPRUCE, N. B. STANDEN, AND P. R. STANFIELD. Multiple blocking mechanisms of ATP-sensitive potassium channels of frog skeletal muscle by tetraethylammonium ions. J. Physiol. Lond. 413: 31-47, 1989. 16. DE WEILLE, J. R., M. FOSSET, C. MOURRE, H. SCHMID-ANTOMARCHI, H. BERNARDI, AND M. LAZDUNSKI. Pharmacology and regulation of ATP-sensitive K’ channels. Pfluegers Arch. 414, Suppl. 1: S80-S87, 1989. 17. FRENCH, R. J., AND J. J. SHOUKIMAS. Blockage of squid axon potassium conductance by internal tetra-n-alkylammonium ions of various sizes. Biophys. J. 34: 271-291, 1981. 18. GELBAND, C. H., N. J. LODGE, AND C. VAN BREEMEN. A Ca2+activated K’ channel from rabbit aorta: modulation by cromakalim. Eur. J. PharmacoZ. 167: 201-210, 1989. 19. HAMILL, 0. P., A. MARTY, E. NEHER, B. SAKMANN, AND F. J. SIGWORTH. Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch. 391: 85-100, 1981. 20. HORN, R., AND K. LANGE. Estimating kinetic constants from single channel data. Biophys. J. 43: 207-223, 1983. 21. Hu, S. L., Y. YAMAMOTO, AND C. Y. KAO. Permeation, selectivity, and blockade of the Ca2+-activated potassium channel of the guinea pig taenia coli myocyte. J. Gen. Physiol. 94: 849-862, 1989. 22. INOUE, R., K. KITAMURA, AND H. KURIYAMA. TWO Ca-dependent K-channels classified by the application of tetraethylammonium distribute to smooth muscle membranes of the rabbit portal vein. Pfluegers Arch. 405: 1.73-179, 1985. 23. KL~CKNER, U., U. TRIESCHMANN, AND G. ISENBERG. Pharmacological modulation of calcium and potassium channels in isolated vascular smooth muscle cells. Arzneim. Forsch. Drug Res. 39: 120126,1989.
MUSCLE
Kca CHANNELS
24. KOVACS, R. J., Y. HUANG, J. BRAYDEN, AND M. T. NELSON. Block of cromakalim action and arterial smooth muscle ATP-sensitive K+ channels by tetrapentylammonium ions (Abstract). Circulation 82: 111-341, 1990. 25. LATORRE, R. The large calcium-activated potassium channel. In: Ion Channel Reconstitution, edited by C. Miller. New York: Plenum, 1986, p. 431-467. 26. LATORRE, R., C. VERGARA, AND C. HIDALGO. Reconstitution in planar lipid bilayers of a Ca2’ -dependent K+ channel from transverse tubule membranes isolated from rabbit skeletal muscle. Proc. Natl. Acad. Sci. USA 77: 7484-7486, 1982. 27. MCCANN, J. D., AND M. J. WELSH. Calcium-activated potassium channels in canine airway smooth muscle. J. Physiol. Lond. 372: 113-127,1986. 28. NEHER, E., AND J. H. STEINBACH. Local anaesthetics transiently block currents through single acetylcholine-receptor channels. J. Physiol. Lond. 277: 153-176, 1978. 29. OGDEN, D. C., AND D. COLQUHOUN. Ion channel block by acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction. Proc. R. Sot. Lond. B Biol. Sci. 225: 329-355, 1985. 30. QUAST, U., AND N. S. COOK. In vitro and in vivo comparisons of two K+ channel openers, diazoxide and cromakalim, and their inhibition by glibenclamide. J. Pharmacol. Exp. Ther. 250: 261271, 1989. 31. QUAST, U., AND C. WEBSTER. Sulfonylureas and tetraalkylammonium ions as inhibitors of cromakalim-stimulated “Rb+ efflux (Abstract). Naunyn-Schmiedebergs Arch. Pharmakol. 337, Suppl: R64, 1989. 32. QUAYLE, J. M., N. B. STANDEN, AND P. R. STANFIELD. The voltage-dependent block of ATP-sensitive potassium channels of frog skeletal muscle by caesium and barium ions. J. Physiol. Lond. 405: 677-697, 1988. 33. SINGER, J. J., AND J. V. WALSH. Characterization of calciumactivated potassium channels in single smooth muscle cells using the patch clamp technique. Pfluegers Arch. 408: 98-111, 1987. 34. SPRUCE, A. E., N. B. STANDEN, AND P. R. STANFIELD. The action of external tetraethylammonium ions on unitary delayed rectifier potassium channels of frog skeletal muscle. J. Physiol. Lond. 393: 467-478, 1987. 35. STANDEN, N. B., J. M. QUAYLE, N. W. DAVIES, J. E. BRAYDEN, Y. HUANG, AND M. T. NELSON. Hyperpolarising vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle. Science Wash. DC 245: 177-180, 1989. 36. STANFIELD, P. R. Tetraethylammonium ions and the potassium permeability of excitable cells. Rev. Physiol. Biochem. Pharmacol. 97: l-67, 1983. 37. VILLAROEL, A., 0. ALVAREZ, A. OBERHAUSER, AND R. LATORRE. Probing a Ca2’ -activate K+ channel with quaternary ammonium ions. PfZuegers Arch. 413: 118-126, 1988. 38. WINQUIST, R. J., L. A. HEANEY, A. A. WALLACE, E. P. BASKIN, R. B. STEIN, M. L. GARCIA, AND G. J. KACZAROWSKI. Glyburide blocks the relaxation response to BRL 34915 (cromakalim), minoxidil sulfate and diazoxide in vascular smooth muscle. J. Pharmacol. Exp. Ther. 248: 149-156, 1989. 39. YELLEN, G. Ionic permeation and blockade in Ca2+-activated K’ channels of bovine chromaffin cells. J. Gen. Physiol. 61: 357-359, 1984.
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ions
P. D. LANGTON, M. T. NELSON, Y. HUANG, AND N. B. STANDEN Department of Physiology, University of Leicester, Leicester LEl 9HN, United Kingdom; and Department of Pharmacology, University of Vermont, Burlington, Vermont 05405
LANGTON, P. D., M. T. NELSON, Y. HUANG, AND N. B. STANDEN.Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium ions. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H927-H934, 1991.The effects of tetraethylammonium ions (TEA+) and tetrapentylammonium ions (TPeA’) on Ca2+-activated K’ (Kc,) channels were studied in membrane patches from mesenteric arterial myocytes. External TEA+ produced a flickery block. The concentration dependence for reduction in mean unitary current was consistent with 1:l binding, with dissociation constants (&) in rat and rabbit of 196 and 159 PM at 0 mV, and the block was weakly voltage dependent. Rate constants for blocking and unblocking were 380 rnM-l= ms-1 and 73 ms-l, respectively. In patches containing several channels TEA+ reduced average current to the same extent as mean unitary current, implying that TEA+ block is independent of the channel state. Block was unaffected by raising external K’ to 120 mM. External TPeA’ blocked with slower kinetics and lower affinity than TEA+ (&, 1.49 mM). The sulfonylurea glibenclamide (lo-100 PM), the hyperpolarizing vasodilator cromakalim (5 PM), and internal ATP (1 mM) were without effect on channel activity. We conclude that TEA+ is a relatively effective blocker of single K ca channels of arterial smooth muscle and should block macroscopic currents equally well, whereas external TPeA+ is about eight times less effective. smooth muscle; tetrapentylammonium; alim
glibenclamide;
cromak-
LARGE-CONDUCTANCECa2+-activated K+ channels (hereafter called Kc, channels) are widespread in smooth muscle and appear to occur at high density in the cell membrane of this tissue (5, 9, 21, 27, 33). Tetraethylammonium ions (TEA+) block many different types of K+ channels with varying degrees of effectiveness (36). External TEA+ is relatively effective in reducing the unitary current amplitude of Kca channels of mammalian muscle and chromaffin cells, with a dissociation constant (KJ in the range 120-300 PM (2, 6, 22, 26, 37, 39), whereas internal TEA+ is much less effective (4, 6, 7, 22). However, although it is widely accepted that TEA+ is an effective blocker of Kca channels in smooth muscle, the stoichiometry, kinetics, and voltage dependence of the blocking reaction of TEA+ are not known. We have therefore characterized the effects of TEA+ on Kca channels of arterial smooth muscle in detail, using patches excised from cells isolated from small branches of rat and rabbit mesenteric arteries. We have investigated the concentration and voltage 0363-6135/91
$1.50
Copyright
dependence and kinetics of block and show that TEA+ block does not lock the channel gate in the open state as happens in ATP-sensitive K+ (KATP) channels of skeletal muscle (15), so that for K ca channels block of macroscopic current should be as effective as that of unitary current. We have also investigated the effects of the larger quaternary ammonium ion tetrapentylammonium (TPeA+) and the sulfonylurea glibenclamide, both of which are very effective at antagonizing the actions of the K+ channel-opening drug cromakalim on vascular smooth muscle. We find that TPeA’ is a less effective blocker of Kc, channels than is TEA+, whereas we could not detect any block by glibenclamide. Cromakalim and internal ATP did not affect channel activity. METHODS Preparation. Rats were killed by stunning followed by decapitation. Rabbits were anesthetized with pentobarbital sodium (35 mg/kg iv) and then exsanguinated. Vascular myocytes were isolated from mesenteric arterial branches 200-300 pm in diameter by enzymatic dissociation. A section of mesenteric artery was removed into cold “isolation buffer” of the following composition (in mM): 140 NaCl, 5 KCl, 1 ethylene glycol-bis@-aminoethyl ether)-N,N,N’,N’-tetracetic acid (EGTA), 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), pH 7.4, adjusted with NaOH. After 30 min the adherent fat was dissected away, and a section with two or three side branches was removed. This tissue was allowed to rest for 15 min in “digest buffer” of the following composition (in mM): 128 NaCl, 5.4 KCl, 4.16 NaHC03, 0.35 Na2HP04, 0.45 KH2PO+ 10 glucose, 2.9 sucrose, 10 HEPES, pH 7.3 with NaOH. The tissue was then tied onto a glass cannula and placed in the barrel of a glass syringe containing 2 ml of the enzyme cocktail. This consisted of collagenase (Sigma Chemical, type IA, 725 U/ml), hyaluronidase (0.3 mg/ml), elastase (Sigma, type IIA, 0.3 mg/ml), and bovine serum albumin (Sigma 7906, 2.5 mg/ml) in digest buffer. The artery was perfused at -0.06 ml/min, and the perfusion solution was collected from the syringe barrel and returned to a reservoir, whence it was recirculated. Both the reservoir and syringe were immersed in a water bath at 35°C. After 20 min, 1 ml of enzyme buffer containing 5 mg trypsin (Sigma, type III) was added, and the tissue was removed 15-20 min later. The dead time from the reservoir to the syringe was 7 min.
0 1991 the American
Physiological
Society
H927
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on October 27, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
H928
TEA+
BLOCK
OF
ARTERIAL
SMOOTH
The tissue was removed, and the side branches were cut off. These were gently triturated in isolation buffer until cells could be observed. The solution was then discarded and replaced by three 2-ml changes of digest buffer. The tissue was triturated in the third aliquot until many cells were obtained. Cells were filtered through a 7-mm filter media mat (RS components) to remove tissue debris and plated into 35mm Petri dishes containing 1 ml of digest buffer. Cells were stored at 4°C and used within 24 h (usually within 8 h). Recording methods. Single ionic channels were studied using the inside-out and outside-out configurations of the patch-clamp technique (19). Patch pipettes were pulled from 1.5 mm OD borosilicate capillaries, coated with Sylgard elastomer, and fire polished. Their resistance was in the range 5-20 MQ when filled with electrolyte. Seals, formed by application of negative pressure, were in the order of 10 Gst. Patches of membrane were excised into the standard inside- or outside-out configuration. Single channels were recorded using a List EPC-7 or Axopatch 1C amplifier. Steady holding potentials, and in some cases step command potentials, were applied to the pipette. According to the usual convention, membrane potentials are expressed inside relative to outside, and outward currents are given a positive sign. The solution bathing the cytoplasmic face of inside-out patches or the external face of outside-out patches could be changed by placing them in the flow from a perfusion pipette consisting of a common outlet connected to four different reservoirs (8). Solutions. For experiments, cells were maintained in conventional physiological salt solution containing (in mM) 137 NaCl, 5.4 KCl, 0.44 KHZP04, 0.42 NaH2P04, 4.17 NaHC03, 11.1 glucose, 1.0 MgC12, 1.8 CaC12, 0.05 EGTA, 10 HEPES-NaOH, pH 7.4. Once seals had formed the bath solution was exchanged for low-calcium isolation buffer to reduce the incidence of vesicle formation on excision. For most experiments the patch membrane was exposed to a near physiological K+ gradient, the composition of the two solutions being (in mM) 1) external: 115 NaCl, 5 KCl, 5 EGTA, 10 HEPES, pH 7.4 with NaOH; and 2) internal: 1 K2ATP, 5 EGTA, 10 HEPES-KOH, KC1 to bring [W] to 124 n&l, pH 7.2. The calculated free [Ca”‘] was ~110~~’ M. A variant of this solution contained 4.13 mM CaC12, giving a free [Ca”‘] of 10m6 M. KZATP was added from a fresh, pHadjusted stock solution on the day of the experiment, and reduced the probability of KArP channel openings occurring. For experiments in which symmetrical high K+ solutions were used, both external and internal solutions contained the following (in mM): 91 KCl, 29 KOH, 5 EGTA, 2.93 CaCIZ, 10 HEPES, pH 7.2, giving free [Ca”] of 2 x 10m7 M. TEA Cl or TPeA Cl was added to external solution at concentrations between 50 PM and 2 mM. Glibenclamide was dissolved at 2 mM in ethanol or at 4 mM in a 50:50 mixture of dimethyl sulfoxide (DMSO) and polyethylene glycol (PEG, mol wt 200). These stocks were added to the internal or external solution to give the desired concentration. Cromakalim (BRL 34915) was usually made up as a 2 mM stock in ethanol or at 10 mM in a 50:50 mixture of
MUSCLE
Kca
CHANNELS
DMSO and PEG and then finally into external solution at the desired concentration. The vehicle concentration was usually ~0.5% and did not cause detectable effects on channel activity (n = 4). Data collection and analysis. Single-channel currents were recorded onto FM or video tape with a recording bandwidth of either 20 or 40 kHz. For analysis the tape was replayed through an eight-pole Bessel filter and digitized with a CED 502 or Labmaster DMA TL-40 Ato-D converter and stored on a 286 or 386 PC or a PDP 11/73 computer. The filter cutoff (-3 dB) frequency and digitization rate were normally 3 and 10 kHz, respectively. Mean unitary currents (mJ in the presence of TEA+ were measured by averaging the digitized record, using cursors to select open periods. For control records, amplitudes measured in this way were the same as those measured by fitting Gaussian curves to amplitude histograms. The kinetics of Kca block by external TEA+ were investigated by the method of amplitude distribution analysis (39) as described previously (32). Open times were measured using cursors spaced midway between current levels. In patches containing up to six channels mean open time (&J was calculated by measuring the times (tj) spent at current levels corresponding to j = 1, 2 N channels open, when the maximum likelihood estimate for & is given by (cjN,ltjj)/nC, where n, is the total number of closings (20). To estimate relative channel activity under different conditions, N Popen, where P openis open-state probability, was calculated as ( &E1 tjj)/ T, where T is the duration of the recording. To measure the effect of blocking agents on the mean current in outside-out patches with several Kca channels we measured the mean current directly from the digitized record. Results are expressed as means t SE, and all experiments were performed at room temperature (18-21°C). l
RESULTS
Flickery block by external TEA+. Block by external TEA+ was studied in inside-out patches so that we could change the solution bathing the internal face of the patch to check that the channels were Ca2+ sensitive. TEA+ was included in the pipette solution at 50, 100, 200, or 500 PM. Large-conductance Kca channels were identified by their conductance and voltage and Ca2+ sensitivity. In the absence of TEA+ and in 5 mM extracellular K’/ 124 mM intracellular K+ concentrations, Kca channels had a slope conductance between -10 and +10 mV of 135 pS (Fig. lB), in good agreement with values reported previously in vascular smooth muscle (5). In each patch Ca2+ sensitivity was tested by switching the flow solution bathing the internal face of the membrane to change [Ca”‘] between 10s6 and 10-l’ M (Fig. 1C): only patches in which channel Popen responded rapidly to intracellular Ca2+ concentrations were used. Many patches contained more than one channel, and in these patches we achieved the periods of single-channel activity needed for analysis of the TEA+ block of unitary currents by mixing 10m6 and lo-” M Ca2+ solutions in the flow pipette, adjusting their flow rates to obtain the desired level of activity. Figure 1A shows unitary currents recorded from Kc,
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TEA+
BLOCK
PM TEA
’
-20
Membrane pA
C [Cd’+],
500
”
-40 3.67
-I ms 2 PA
SMOOTH
-*
1 Ow6 M
”
1’
*
4
0
potent%
(n$
d”td &jM _- - - - - -/I. - - - - - -c---
/Al TEA 1.52 C
pA
FIG. 1. External tetraethylammonium ions (TEA+) block of Ca”activated K+ (Kc,) channels. A: recordings from 3 inside-out patches from rat mesenteric arterial myocytes held at 0 mV. Pipette solution contained no TEA+ (top), 100 ,uM TEA+ (middle), 500 PM TEA+ (bottom). C, closed level in each case; broken line, mean amplitude of unitary current; filter, 3 kHz; sample rate, 10 kHz. B: unitary currentvoltage relationship in the absence (a) and presence (A) of 500 FM TEA+. Each point is mean t SE, where this is larger than symbol from 5 patches. Line through control points is drawn by eye and was scaled to fit points in 500 PM TEA+. C: Ca2+ dependence of channel activity in inside-out patch with several channels. Patch was held at 0 mV, and pipette contained 50 ,uM TEA+. Flowing solutions bathing internal face of patch were switched between 10v6 M Ca2’ (solid line) and 10-l’ M Ca” (dashed line). For display, record was filtered at 1 kHz.
channels in a patch in the absence of external TEA+ and from patches in which the pipette solution contained TEA+ at 100 and 500 PM. It can be seen that TEA+ caused a concentration-dependent reduction in the mean single channel current and increased the open channel noise. The rni amplitude at 0 mV was reduced from 5.38 t 0.11 pA (n = 5, rat mesenteric arterial myocytes) in control to 3.67 t 0.13 pA (n = 4) in 100 PM TEA+ and 1.54 t 0.07 pA (n = 5) in 500 PM TEA+. The mean current-voltage relationship in control patches and in patches with 500 FM external TEA+ is shown in Fig. 1B. This type of flickery block is characteristic of a blocking process with relatively rapid kinetics, so that blocking and unblocking events are not fully resolved at the recording frequency used (3 kHz in the case of Fig. 1). Thus a flickery open period in the presence of TEA+ is composed of times spent in both the open and blocked states. The block can be considered in terms of the two general types of blocking model given below CT
k1 k -1
cc,O)’
kb[TEA+]
‘OK
\ l-3
k -b
kb[TEA+]
k
MUSCLE
H929
Kca CHANNELS
(i) will be given by
1.
10
ARTERIAL
B ‘9
Control
100
OF
(scheme 1)
Figure 2A shows the dependence of mJi on TEA+ concentration at 0 mV. The points are well fit by Eq. 1, consistent with 1:l binding of TEA+ to a receptor and with a & (fitted by a least-squares algorithm to results from 18 patches) of 196 PM. The mean of the & values for individual patches from rat myocytes at 0 mV was 193 t 9 PM (n = 18), whereas the value for rabbit myocytes of 159 t 34 PM (n = 9) was not significantly different (P = 0.22, unpaired t test). It can be seen from Fig. 1B that the voltage dependence of the block must be very slight, as the mean current-voltage relationship in 500 PM TEA+ is quite well fit by scaling the control relation. & values were measured at membrane potentials between -40 and +40 mV by fitting m;/i as described above. The dependence of & on membrane potential is shown in Fig. 2B. The line shows a least-squares fit to the Boltzmann equation & = & (0) exp (z 'VF/ RT) with J&(O), the & at 0 mV = 196 ,uM, giving an equivalent valency for the block (x ‘) of 0.06. This suggests that the block by TEA+ shows very slight voltage dependence in the direction expected for an external cation, increasing with hyperpolarization, but that the ion must experience only very little of the membrane voltage field. Kinetics of TEA+ block. Two approaches have been used to extract rate constants from partially resolved blocks of the type seen here. The distribution of current amplitudes in the presence of blocker may be fitted with a ,&function convolved with the Gaussian distribution of the closed level current, varying the block and unblock rates to obtain the best fit (32, 39). Alternatively, the analysis may be done in the frequency domain, by measuring the variance from the difference power spectrum (open minus closed level) of the current noise in the presence of blocker. The ratio of the variance calculated assuming that the blocker causes brief complete channel
A
B 1.0 0.8
\ l
-
0.6 -
i-
0.4 -
1ooJ -40
Membrane
L (C, 0)B
(scheme2)
-b
where C represents closed states of the channel, 0 represents open states, and B represents blocked states. In scheme 1 the blocker binds to the open state and must unbind before the channel can close, whereas in scheme 2 block is independent of the channel state. In each case kb and k-b are the rate constants for blocking and unblocking, so that Kd = k-b/kb. For either scheme, rn; in the presence of TEA+ as a fraction of its control value
-20
0
potential
20
40
(mV)
FIG. 2. Concentration and voltage dependence of TEA’ block in rat arterial myocytes. A: plot of fractional mean unitary current (mJi), at 0 mV, against blocker concentration. Points are means t SE, where larger than symbol from 4-6 patches. Curve is drawn to Eq. 1 which assumes 1:l binding with dissociation constant (J&) of 196 PM. B: voltage dependence of & (e) values from reduction in m;/i (each point is a least-squares fit to results from 14-20 patches, except for that at -40 mV, which is from 3 patches). A, values obtained as rate constants for unblocking and blocking (h-&b) from amplitude distribution analysis (mean t SE of 6-14 values at each voltage). Line is least-squares fit to the Boltzmann function given in text with &(O) = 196 PM and equivalent valency (z ‘) = 0.06.
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H930
TEA+
BLOCK
OF
ARTERIAL
SMOOTH
closings to the measured variance is then used to estimate the corner frequency of the blocking process (29, 32). In the present study, we have concentrated on the amplitude distribution method because this is easier to apply to data in which single channel openings may be quite brief and also because the method gives estimates for both blocking and unblocking rates, allowing calculation of the &, which may then be compared with that measured independently from the reduction in rn; (Fig. ZB). In cases where we were able to use the excess variance method, this gave similar values for the rates. Figure 3 shows current amplitude histograms in a control patch (Fig. 3A) and in a patch exposed to 500 PM TEA+ (Fig. 3B). In the absence of TEA+ both the closed and open current peaks may be well fit with Gaussian distributions. In 500 PM TEA+ the open level peak is moved to a much lower amplitude and the distribution is skewed so that it is no longer Gaussian. Instead, the smooth line fitted to the open level shows a ,& distribution generated using a blocking rate (= hb[TEA’]) of 162 ms-1 and an unblocking rate, h-b, of 57 ms-‘. The blocking rates measured as described above increase with [TEA+] (Fig. 4A), as expected from the models presented in schemes 1 and 2, and the slope of the relationship (which gives the rtzb)is greater at 0 than +40 mV. The k -b did not change significantly with [TEA+], for example, it was 71 t 4 (n = 11, rat myocytes) in 100 PM TEA+ and 65 t 4 (n = 10) in 500 PM TEA+ (P = 0.20, unpaired t test). The dependence of the rate constants kb (calculated as kb/ [TEA’] ) and k-b on voltage (Fig. 4B) shows that the observed small voltage dependence of the Kd appears to reside entirely in the voltage dependence of the blocking reaction. These rate constants were also used to calculate values for & at different voltages. Figure 2B shows that these values agree well with those from measurements of the reduction in Illi.
MUSCLE
Kca CHANNELS
A -
B 250
-iV-I 200 E -
2 ,o
I
fj/y
600 500 400 300
~:::~,~~~,~
0.1
0.3 0.5 [TEA+] (mM)
-20 Membrane
0
20
40
potential
(mV)
FIG. 4. A: dependence of block rate on [TEA’] at 0 mV (0) and +40 mV (A). Points show means (*SE where larger than symbol) of measurements from 11-14 patches. Straight lines are least-squares fits giving blocking rate constants of 399 and 268 mM-‘.rns-’ at 0 mV and +40 mV, respectively. B: rate constants for blocking (kb) (A, units rnM-‘* ms-‘), and unblocking (k-b; l , U/ms-‘) as functions of voltage. Points show means & SE from II-20 patches of rat myocytes.
A Control _.-- I -!ivvq
-.- . , -I- - - -
----- I control
P
= 6.37
pA
C
B ----- ‘TEA+ C
1.0 r
= 3.15
pA
5 pAI f-
10:s -b
\ I-
% F
*
0.4 I 0.2
t
OOL .
A 50
I 100
‘
200
[TEA+1WI 5. TEA’ block in outside-out patches. A: recording from patch held at 0 mV and exposed to control (TEA+-free) external solution. Continuous line shows closed level (c) and broken line mean current level. B: record from same patch in 200 PM external TEA+. C: comparison of reduction in unitary and overall mean current by TEA’. Open bars show m;/i replotted from Fig. 3, whereas solid bars show average current in presence or absence of TEA+ (&rEA+JzontrOJ measured over lo-30 s in outside-out patches like that of A and B above (mean t SE of 4 patches of rat myocytes). FIG.
A 3
B
Control
c
500pM
TEA+
01
0 Current
(PA)
Current
2
4
6
(PA)
FIG. 3. Amplitude distribution analysis of TEA+ block in rat arterial myocytes. A: histogram of current amplitude from patch in absence of external TEA+ held at +20 mV. C and 01, closed and open current levels; curves are Gaussians fitted to these peaks, giving unitary current amplitude of 8.90 pA. B: amplitude histogram from patch with 500 PM external TEA+ and also held at +20 mV. Closed current level is fitted with Gaussian curve, but broadened and skewed open level peak has been fitted by eye with ,&distribution of form f(y) = y’“-I’( 1 - y)(“-‘I/ B(a,b) where B(a,b) = JAy’“-“(l - y)@-l’dy, a = k-b7, b = kb[TEA+]T, and 7 is 0.228/fc, where fc is -3 dB frequency of &pole Bessel filter (1 kHz in this case), convolved with baseline Gaussian (32). Fit gave block rate as 162 ms-’ and unblock rate as 57 ms-‘.
Block of mean currents in outside-out patches by TEA 7 The two types of blocking scheme above have different consequences for the relationship between block of unitary current and the effect of the blocker on macroscopic currents. If block prevents channel closure as in scheme 1, the mean length of an open-blocked burst will increase with blocker concentration, with the total & per burst remaining constant (14,28). The increase in burst length will compensate for the reduction in mean current during the burst, and as a consequence the block of macroscopic current will be slight when Popenis low. The &s for reduction in m; and for block of macroscopic current (& and Ki), respectively, will be related by Ki = KJP,,,,, so block of macroscopic current only becomes as effective as that of unitary current as Popenapproaches unity (15). For the state-independent block of scheme 2, in contrast,
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TEA+
BLOCK
OF
ARTERIAL
SMOOTH
the blocker will reduce unitary and macroscopic currents equally well, irrespective of Popen. External TEA+ block of K+ channels may be of either type: in frog skeletal muscle that of delayed rectifier channels is state independent, whereas TEA+ block of KATP channels presents channel closure (15, 34). We investigated therefore which type of scheme is appropriate for TEA+ block of Kca channels. Patches were excised in the outside-out configuration, using a constant [Ca”‘] of 10B6 M in the internal solution. Such patches normally had several active Kca channels, and we measured the mean current flowing through the patch in different external TEA+ concentrations (Fig. 5). Mean current was measured over periods of lo-30 s. In four patches, channel Popen in control was 0.20,0.41,0.42, and 0.65, so that Ki should have been significantly higher than & for a block of the type in scheme 2. In fact, TEA+ reduced the mean current in outside-out patches by the same amount as it did rni (Fig. 5C). We conclude that the block in this case is of the type shown in scheme 2, and does not prevent channel closure, so that TEA+ should be as effective in blocking macroscopic Kca. currents as it is in blocking single channel currents. TEA+ block in high external K+. Raised external K+ enhances block of Kca channels by external Cs+ (12) but relieves block by internal TEA+ (37). We therefore looked for possible changes in block by external TEA+ when outside-out patches from rat arterial myocytes were bathed in symmetrical 120 mM K+ solutions. Under these conditions, the unitary current-voltage relationship was linear with a slope conductance of 288 pS. Each patch was exposed to TEA+-free solution and to 50, 100, 200, and 500 PM TEA+. Kd values were measured from mi/i and were 164 t 9 ,uM (5 patches, rat myocytes) at -20 mV and 229 t 17 PM (4 patches) at +20 mV. Neither Kd was significantly different from that measured at the
MUSCLE
Kca
CHANNELS
H931
same voltage in 5 mM external K’; these were 172 t 14 PM (14 patches) at -20 mV and 227 t 15 PM (18 patches) at +20 mV (P = 0.73 and 0.97, respectively, unpaired t test). Thus we found no evidence for either enhancement or relief of external TEA+ block by high external K+. Effect of TPeA+. In addition to TEA+, other quaternary ammonium ions block K+ channels, either more or less effectively (17, 36). We investigated the effect of TPeA+ because this has been shown to be a potent blocker of the efflux of 86Rb+ induced by the K+ channel opener cromakalim in rat aorta (31). TPeA+ was applied to the external face of outside-out patches from rat arterial myocytes at concentrations of 0.2, 1.0, and 2.0 mM. Figure 6A shows that TPeA+ blocks with slower kinetics than ‘TEA+, causing a concentration-dependent reduction in open time. The concentration-effect curve for the reduction in mean current by TPeA+ is shown in Fig. 6B and is reasonably well fit assuming 1:l binding with a Kd of 1.49 mM. If an open channel can leave the open state by two routes, closing or blocking, the & will be given by the reciprocal of the sum of the rates for these routes (see, e.g., Ref. 13), so that here & = l/ (kBl + kb[TPeA+]), where k-, is the rate constant for channel closing. Thus l/i0 should be a linear function of [TPeA+]. We did not study the kinetics of TPeA+ block in detail, but the results from two patches, shown in Fig. 6C, suggest that this is so. The slope indicates a kb of 0.36 rnM-‘* ms-‘, and the unblocking rate constant, estimated as kb Kd is 0.54 ms? Effects of glibenclamide, internal A TP, and cromakalim. A widespread feature of the effects of K+ channel opening drugs such as cromakalim is that they are blocked by glibenclamide, a sulfonylurea thought to be specific for KATP channels (1, 16). Glibenclamide is lipid soluble and appears to act when applied to either face of the membrane (1). Because cromakalim has been reported to l
A
cc
Control *
0.8
-
,.! \ I-
0.6
-
0.4
-
0.2
t
.
0.01 0.100
1 .O mM TPeA+
[TP~A+]
e
0.8
; w
0.6
,+o
0.4
\ -
0.2
.
1.000
(mM)
2.0 mM TPeA+
5 PA I 10 ms
0.0”“““““““““““”
FIG. 6. Block by tetrapentylammonium ions (TPeA’). A: records from outside-out patch held at 0 mV and with its external face exposed successively to TPeA+-free solution and to solutions with TPeA’ at 0.2, 1.0, and 2.0 mM. Filter 3 kHz; sample rate, 10 kHz. B: reduction in mean current by TPeA’. Points show average current in presence or absence of TPeA+ ( &TreA+&,ntrol; mean t SE, 4 patches. Line is drawn to Eq. 1 with & = 1.49 mM. C: plot of reciprocal mean open time against [TPeA’]. Symbols show results from 2 different patches of rat myocytes and slope of line gives hb as 0.36 mM-’ ms? l
1 .o
[TP~A+]
2.0
(mM)
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on October 27, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
H932
TEA+
BLOCK
OF
ARTERIAL
SMOOTH
MUSCLE
Kca
CHANNELS
A Glibenclamide
(15pM)
1 OOnM Calcium
1
+ *M
I
Cromakalim
III
-
2OpAL
+ 6.wM
Calcium
II
( patch
potential
= +40mV)
Ssec
C
1 mM ATP
B 0.5 lOPAl
0.4
30sec 12
T
I
k 0.3
0
g 0.2 0.1 Current
(PA)
Current
activate Kca channels of smooth muscle (l&23), we have looked for blocking effects of glibenclamide on Kc, channels. In inside-out patches excised from rabbit mesenteric arterial myocytes we could not detect any difference in activity measured as No Popenbetween the absence and presence of glibenclamide at concentrations of lo-20 PM (P = 0.65, paired t test, 8 patches, Fig. 7). In the same patches, glibenclamide did not affect unitary current amplitude, which was 5.44 t 0.34 pA in control and 5.48 + 0.38 pA in glibenclamide (P = 0.64, paired t test). Similarly, in three outside-out patches from rat artery, we detected no effect of external glibenclamide. Mean values were 2.98 t 0.67 in control and 3.01 t 0.66 in 50 PM (2 patches) or 100 PM (1 patch) glibenclamide (P = 0.12, paired t test). Because cromakalim and glibenclamide have been shown to affect K ATp channels in arterial smooth muscle and other tissues (1, 35), we investigated the possibility that there may be functional similarity between KATP and Koa channels by testing the effects of internal ATP (1 mM) on Kca channels (Fig. 7). ATP did not affect the unitary current or Popenof Kc, channels. In six patches N
l
Popen
0
7. A: recording
from inside-out, patch from rabbit mesenteric arterial myocyte held at 0 mV showing effect of 15 FM glibenclamide. B: effects of glibenclamide on 8 inside-out patches as in A above. Activity, measured as N. Popen,was measured over periods of 5-8 min. Open bar shows mean control activity -I- SE and hatched bar shows mean activity in lo-20 PM glibenclamide (3 patches were exposed to 10, 4-15, and l-20 PM). C: lack of effect of internal ATP on Kca channels from rabbit mesenteric artery. Original records with amplitude histograms from longer segments of recordings are shown. N. Popen before and after ATP (as Na2ATP) was 1.80 and 1.77. Internal-free Ca”+ was 6.5 FM and patch potential was 0 mV. The 1 mM ATP was calculated to lower Ca2+ under these conditions from 6.5 to 5.1 PM. Apparently long channel openings displayed in Figs. 7 and 8 are due to program used to plot long segments of record, which leaves out brief closings. FIG.
0
(PA)
+20 +40 +60 Membrane Potential, mV
n O.lpMca 0
gx1 0.1 pM Ca, 5 PM Cromakalim
6.5 PM Ca, 5 FM Cromakalim
FIG. 8. Lack of effect of cromakalim on Kca channels at 4 different voltages. A: recording from inside-out patch from rabbit mesenteric arterial myocyte held at +40 mV, in 0.1 PM Ca2+, in 0.1 PM Ca2+, and 5 PM cromakalim, and in 6.5 PM Ca2+ and 5 PM cromakalim. N-Pop,, of Kca channels in these 3 solutions was 0.005, 0.003, and 2.01, respectively. B: effects of cromakalim (5 FM) on N. Popenat 0, +20, +40, and +60 mV (n = 6 patches). For convenience of display, error bars are not shown at 0 and +20 mV. NoPop,, at 0 mV with and without cromakalim were 0.003 & 0.0008 and 0.002 & 0.0008. N. Popenat +20 mV with and without cromakalim were 0.017 * 0.007 and 0.012 t 0.005. To verify its activity, cromakalim used in each experiment on Kca channels was shown to relax norepinephrine (10 PM)-constricted mesenteric arteries, with ~50 nM cromakalim causing full relaxation.
from rabbit myocytes No Popenwas 1.55 t 0.34 in control and 1.55 t 0.33 in 1 mM internal ATP. We also investigated the effects of cromakalim itself on six patches excised from rabbit arterial myocytes. The cytoplasmic face was exposed to low7 M Ca2+, and cromakalim (5 PM) was also applied to this face. Cromakalim caused a small reduction in channel Popenat all voltages tested (Fig. 8), which was not statistically significant (P > 0.05, paired t test). In three patches from rat myocytes we found no effect of 10 PM cromakalim on channel activity. DISCUSSION
Properties of block by external TEA+. TEA+ produced a flickery block of unitary currents through Kca channels. The & values for the reduction in rni of 196 ,uM in rat and 159 PM in rabbit agree well with those for external TEA+ block of Kca of rat myotubes and chromaffin cells that are in the range 100-300 PM (6, 26, 37, 39). In
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TEA+
BLOCK
OF
ARTERIAL
SMOOTH
smooth muscle & values of 300 PM have been reported in rabbit portal vein (2) and of 100-300 PM in taenia coli (21). We find a 1:l stoichiometry for TEA+ binding, as for block of delayed rectifier and KATP channels by external TEA+ (15, 34). The weak voltage dependence that we find, corresponding to an z’ of 0.06, also seems to be generally true for K+ channel block by external TEA+, with z’ values in the range O-0.2, so that TEA+ seem to enter only a little way into the membrane voltage field to reach its blocking site (6, 15, 34, 37, 39). The & for block by external TEA+ was unaffected by raising external K+ from 5 to 120 mM. Raised external K+ has been reported to enhance the block by external Cs+ of Kea channels reincorporated from rabbit intestinal smooth muscle into lipid bilayers (12). The Cs+ block is strongly voltage dependent, and the enhancement is consistent with a multi-ion pore model for the channel in which K+ can enter behind a blocking Cs+. The weak voltage dependence of block by external TEA+ indicates a much more superficial binding site, preventing a similar effect of external K+. Raised external K+ has also been found to reduce block by internal TEA+ in reincorporated channels from t tubules of skeletal muscle, suggesting competition between K+ and TEA+ for its blocking site (37). The lack of reduction in external TEA+ block by high K+ that we observe exemplifies the difference between the internal and external binding sites for TEA+ (25, 37). Internal block shows greater voltage dependence, and longer chain quaternary ammonium ions are more effective than TEA+ (37). In contrast, the external site is much more specific for TEA+, tetramethylammonium, or tetrapropylammonium for example, being around ~400 less potent (37). We found that external TPeA+ was more effective than these ions, blocking mean current with a & of 1.49 mM, about eightfold higher than that for TEA+. We estimate the rate constants for TEA+ block from ,&function fits to the distributions of current amplitude as 380 t 27 rnM-l ems-’ and 73 t 5 ms-’ for blocking and unblocking, respectively, at 0 mV. TEA+ block of Kca channels of skeletal muscle reincorporated into bilayers has quite similar kinetics, with rate constants of 210 mM-’ l rns-l and 63 ms-’ (37). The kinetics of TEA+ block have also been studied at the single channel level in delayed rectifier and K ATp channels of skeletal muscle. In both these cases TEA+ is much less effective, with & around 7 mM, and the kinetics are faster, so that TEA+ reduces unitary currents without increasing open channel noise (15, 34). TEA+ as blocker of macroscopic Kc, current. Our results show that TEA+ reduction of average current, measured in outside-out patches, follows closely the fractional reduction in unitary current. Because the average current (r) will be given by r = No i. Popen, where i is the unitary current, and Popen is in this case the probability that the channel gate is open, whether or not the channel is blocked by TEA+, the fact that Tand i decline in proportion implies that N* Popen does not change. This suggests that at least under our experimental conditions, external TEA+ blocks the channel without interfering with the channel gating mechanism. Thus external TEA+ should be an effective blocker of macroscopic Kc, current and
MUSCLE
Kca
H933
CHANNELS
because of its quite low Kd may be useful in separating effects on Kca from other K+ currents in smooth muscle. The report that 4 mM TEA+ completely inhibited macroscopic Kca current in rabbit portal vein (3) is consistent with this. In contrast, external TEA+ is less effective at blocking macroscopic currents through KATP channels than it is at reducing unitary current because block prevents channel closure (10, 15). Actions of cromakalim, glibenclamide, and TPeA+ on Kc, channels. Although we could not detect activation of Kca channels of mesenteric artery by the K+ channel opener cromakalim, it has been reported to activate Kca channels in patches excised from vascular smooth muscle and after incorporation into bilayers from rabbit aorta, and it has been suggested that activation of these channels may underlie some of the hyperpolarizing and relaxing action of K+ channel openers on vascular smooth muscle (18, 23). However, these actions are relatively insensitive to TEA+ (2, 35, 38). Because external TEA+ is an effective blocker of Kca channels this argues against their involvement in the functional effects of K+ channel openers. Three other blockers have been found to be potent antagonists of the effects of K+ channel openers; glibenclamide (11, 30, 35, 38); TPeA+, which inhibits cromakalim-induced “Rb+ efflux with a Kd of 0.1 PM (31) and completely reverses cromakalim relaxations of mesenteric arterial rings at 10 PM (24); and Ba2+ (35). We examined the effects of two of these agents on Kc, channels. As discussed above, TPeA+ was less effective than TEA+, whereas we could not detect any effect of glibenclamide at concentrations between 10 and 100 PM on Kca channels. This also argues against the involvement of Kca channels in the effects of K+ channel openers on vascular smooth muscle. We thank Dr. J. Hescheler, who took part in some of the early experiments on rabbit cells, Drs. N. W. Davies and J. M. Quayle for discussions and for comments on the manuscript, and W. King for expert technical assistance. The work was supported by grants from the Wellcome Trust, Medical Research Council, North Atlantic Treaty Organization, American Heart Association (AHA), and National Science Foundation. Y. Huang is supported by a fellowship from AHA, Vermont Affiliate. M. T. Nelson is an Established Investigator of the AHA, and N. B. Standen holds a Wellcome Research Leave Fellowship. Cromakalim was a gift of Beecham Pharmaceuticals. Received
4 June
1990; accepted
in final
form
9 October
1990.
REFERENCES 1. ASHCROFT, F. M. Adenosine 5’-triphosphate-sensitive potassium channels. Annu. Reu. Neurosci. 11: 97-118, 1988. 2. BEECH, D. J., AND T. B. BOLTON. Properties of the cromakaliminduced potassium conductance in smooth muscle cells isolated from the rabbit portal vein. Br. J. Pharmacol. 98: 851-864, 1989. 3. BEECH, D. J., AND T. B. BOLTON. Two components of potassium current activated by depolarization of single smooth muscle cells from the rabbit portal vein. J. Physiol. Lond. 418: 293-309, 1989b. 4. BENHAM,~. D.,T. B. BOLTON, R. J. LANG, AND T. TAKEWAKI. The mechanism of action of Ba2’ and TEA on single Ca’+-activated K’-channels in arterial and intestinal smooth muscle cell membranes. Pfluegers Arch. 403: 120-127, 1985. 5. BENHAM, C. D., T. B. BOLTON, R. J. LANG, AND T. TAKEWAKI. Calcium-activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery. J. Physiol. Lond. 371: 45-67, 1986.
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H934
TEA+
BLOCK
OF
ARTERIAL
SMOOTH
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