Journal of Physiology (1992), 450, pp. 469-490 With 7 figures Printed in Great Britain

469

CALCIUM CHANNEL CURRENTS IN XENOPUS OOCYTES INJECTED WITH RAT SKELETAL MUSCLE RNA

BY NATHAN DASCAL, ILANA LOTAN, EHUD KARNI AND ARIELA GIGI From the Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel (Received 9 July 1991) SUMMARY

1. Ba2+ currents (IBa) through voltage-dependent Ca2+ channels were studied in Xenopus laevis oocytes injected with heterologous RNA extracted from skeletal muscle (SkM) of young rats, using the two-electrode voltage clamp technique. 2. With 40 or 50 mM-extracellular Ba2+, native oocytes of most frogs displayed IBa between -5 and -20 nA at 0 mV. However, in 'variant' native oocytes of four frogs, IBa exceeded -30 nA and reached up to -100 nA. In oocytes injected with SkM RNA, IBa of up to -250 nA was observed. 3. In SkM RNA-injected oocytes and 'variant' native oocytes, the decay of IBa displayed two kinetic components. The faster component was selectively blocked by 40-100 guM-Ni2+ and thus was termed the Ni2+-sensitive IBa. The slower component was Ni2+ resistant, being inhibited only 10-20% by 100-200/tM-Ni2+. The halfactivation and the half-inactivation voltages of the Ni2+-sensitive IBa were more negative (by 14-5 and 28-7 mV, respectively) than those of the Ni2+-resistant IBa. 4. Neither Ni2+-sensitive nor Ni2+-resistant IBa in native or SkM RNA-injected oocytes were affected by dihydropyridine antagonists nifedipine and (+) PN 200-110 (1-10 /M), by the dihydropyridine agonist (-)Bay K 8644 (0-01-2 gM), or by verapamil below 50 JLM. IBa was blocked by diltiazem (half-block at about 500 /tM). Thus, the pharmacology of IBa in SkM RNA-injected and in native oocytes was not characteristic of the L-type Ca2+ channel abundant in the skeletal muscle. 5. Destruction of the RNA coding for the channel-forming tx,-subunit of the SkM L-type Ca2+ channel using a hybrid arrest method failed to selectively suppress the appearance of either Ni2+-sensitive or Ni2+-resistant IBa in SkM RNA-injected oocytes. 6. Our results suggest that the appearance of large voltage-dependent Ba2+ currents in SkM RNA-injected oocytes is not due to the expression of the a1-subunit of the SkM L-type Ca2+ channel. The possibility that the expression of a channelforming subunit of another Ca2+ channel type underlies one of these currents cannot be rejected. However, since the Ba2+ currents in SkM RNA-injected oocytes resemble those observed in native oocytes, we suggest that their appearance may be the result of an enhanced activity of the native Ca2+ channels, possibly due to the expression of the 'auxiliary' subunits of the SkM Ca2+ channel that form complexes with a native al-subunit. MS 9539

470

N DASCAL AND OTHERS INTRODUCTION

Members of the diverse group of voltage-dependent calcium channels play an important role in excitation-contraction and excitation-secretion coupling, transmitter release, and cellular excitability. In smooth, cardiac and skeletal muscle (SkM), a transient, low-voltage-activated current (T), and a slower, high-voltageactivated, dihydropyridine (DHP)-sensitive current (L) have been found, the latter being the dominant one (see Bean, 1989). The SkM Ca2" L-channel protein consists of five subunits: a, (- 165 kDa), x2 (- 130 kDa),,8 (f 52 kDa), y (- 33 kDa) and 6 (- 25 kDa) (for review, see Catterall, 1988). al is the main, channel-forming subunit (Tanabe, Beam, Powell & Numa, 1988; Lotan, Goelet, Gigi & Dascal, 1989b; PerezReyes, Kim, Lacerda, Horne, Wei, Rampe, Campbell, Brown & Birnbaumer, 1989). Injection into Xenopus oocytes of cardiac or smooth muscle a cRRNA (RNA transcribed in vitro from cloned cDNA) yielded functional L-type Ca21 channels (Mikami, Imoto, Tanabe, Niidome, Mori, Takeshima, Narumiya & Numa, 1989; Biel, Ruth, Bosse, Hullin, Stuhmer, Flockerzi & Hofmann, 1990), whereas cRNA of a brain ocx-subunit directed a DHP-insensitive Ca21 channel (Mori, Friedrich, Kim, Mikami, Nakai, Ruth, Bosse, Hofmann, Flockerzi, Furuichi, Mikoshiba, Imoto, Tanabe & Numa, 1991). Co-expression of cardiac or brain al-subunit with the 'auxiliary' SkM subunits (fi, a2/& and/or y) enhanced the expression of the channel and affected its properties (Mori et al. 1991; Singer, Biel, Lotan, Flockerzi, Hofmann & Dascal, 1991). Functional SkM L-channels were expressed in a mammalian cell line transfected with cDNAs of al-subunit alone or together with the auxiliary subunits (Perez-Reyes et al. 1989; Lacerda, Kim, Ruth, Perez-Reyes, Flockerzi, Hofmann, Birnbaumer & Brown, 1991; Varadi, Lory, Schultz, Varadi & Schwartz, 1991). The ability to express the Ca2+ channel in the powerful oocyte model system can strongly promote the study of the various molecular aspects of the channel's function. However, attempts to express the SkM al-subunit in the oocyte, with or without the 'auxiliary' subunits, have been unsuccessful (see Dascal, 1990, and our unpublished data). One possibility is that the oocyte lacks a cellular component, possibly an SkM-specific protein; if so, one might expect that the oocyte will express the SkM Ca2+ channel after injection of heterologous SkM mRNA, that contains hundreds of mRNAs coding for SkM proteins. In fact, the oocytes successfully expressed Ca2+ channels after the injection of heterologous RNA from heart (Dascal, Snutch, Lubbert, Davidson & Lester, 1986; Moorman, Zhou, Kirsch, Lacerda, Caffrey, Lam, Joho & Brown, 1987), Torpedo electric organ (Umbach & Gundersen, 1987) and brain (Dascal et al. 1986; Leonard, Nargeot, Snutch, Davidson & Lester, 1987). On the other hand, brain RNA never produced an L-type channel in the oocyte system (Leonard et al. 1987), suggesting that the oocyte is for some reason inadequate for expressing particular Ca2+ channels from some tissues. In preliminary experiments, we frequently observed Ca2+ channel currents in oocytes injected with rat SkM RNA (Dascal et al. 1986; Lotan, Gigi & Dascal, 1989 a). We decided to carefully characterize these currents on the macroscopic level, and to test whether the expressed channel is of the L-type. In this study we found that heterologous SkM RNA directed the expression of two types of Ca2+ channel current in Xenopus oocytes, separable by their sensitivity to block by Ni2+, kinetics,

Ca`2 CHANNELS IN MUSCLE RNA-INJECTED OOCYTES

471

and activation and inactivation properties. However, none of these currents had the pharmacological characteristic of an L-type, and hybrid arrest experiments showed that none of them was encoded by a message corresponding to the SkM L-channel alsubunit. Furthermore, we found that the properties of these currents were very -similar to those of the endogenous ('native') currents observed in oocytes that have not been injected by any RNA. We conclude that most of the expressed Ca2+ channel currents are due to enhancement of the activity or functional expression of a native oocyte's Ca2+ channels, and discuss a possible mechanism by which SkM RNA could cause this effect. METHODS

Oocytes Xenopua laevis females were purchased from the African Xenopua Facility (Fish Hoek, South African Republic) or from Xenopus One Co. (Ann Arbor, MI, USA). The animals were maintained at a 11 :13 h light: dark cycle at 18-21 'C. To harvest oocytes, the frog was anaesthetized with 0 15% MS-222 (tricaine methanesulphonate, Sigma) to full immobility; a small incision was made on the belly, and the desired number of oocytes was dissected out. The incision was then sutured, and the animal returned to the tank and allowed to recover for at least 2 months before the next surgery (for details, see Dascal, Landau & Lass, 1984). If the ovary was devoid of oocytes, the frog was killed by double pithing after being anaesthetized with MS-222. Oocytes (stage 5 and 6) were defolliculated at 22 'C by 3-4 h treatment with collagenase (Sigma Type IA, 1-5-2 mg/ml) in Ca2+free ND-96 solution (Table 1). After defolliculation, the oocytes were washed with normal ND-96 and injected as described previously (Dascal et al. 1986) with either 200-500 ng of total RNA or 100 ng of poly(A)RNA extracted from skeletal muscle of rats (ages 7-30 days). The oocytes were then incubated for 2-8 days in ND-96 solution with the addition of 1-8 mM-CaCl2, 2-5 mM-sodium pyruvate, 100 ,tg/ml streptomycin and 100 units/ml potassium penicillin at 22 'C and then used for the electrophysiological experiments. In most cases, the oocytes were injected with 200-400 pmol ethyleneglycol-bis-(fl-aminoethylether)-N,N,N',N'-tetracetic acid (EGTA), titrated with KOH or NaOH to pH = 7-2, 1 day before the experiment, to prevent fluctuating Cl- currents often caused by the addition of Ni2+ or Cd2+ to the external solutions. These currents resembled the oocyte's muscarinic response (Dascal et al. 1984), and were probably due to the release of Ca2` from an intracellular source. Materials Rats were anaesthetized with ethyl ether and killed by decapitation with a guillotine. Total RNA was extracted from hindlimb SkM using a guanidine hydrochloride procedure modified as described elsewhere (Dascal & Lotan, 1991 b). RNA was dissolved in water at 4-8 mg/ml and stored in 3-10,l aliquots until injection into the oocytes. Total oocyte RNA was prepared from fully defolliculated oocytes using the LiCl-urea procedure (see Dascal & Lotan, 1991 b). Poly(A)RNA was prepared by passing the total RNA through an oligo-dT column as described previously (Maniatis, Fritsch & Sambrook, 1982) and stored as above. Oligonucleotides cDHPR-O1 and cDHPR-02 were the same as used in previous studies (Lotan et al. 1989b). They were hybridized with SkM RNA for 2-4 h at 37 'C (after a 5 min hybridization at 65 °C), as described by Lotan et al. (1989b), before injection into the oocytes. The hybridization buffer contained 200 mM-NaCl and 10 mMTris-HCl (pH = 7 4). All chemicals used for preparation of RNA were of molecular biology grade, except guanidine hydrochloride which was of analytical grade. Phenol was purchased as analytical grade crystals from Merck (Darmstadt, Germany), and distilled in our laboratory. The reagents used in electrophysiological experiments were of analytical grade. All chemicals listed below were from Sigma (St Louis, MO, USA). Tetrodotoxin (TTX), 3',5'-cyclic adenosine monophosphate (cyclic AMP; sodium salt) and diltiazem were dissolved in water at 1, 10 and 10 mM, respectively, and stored at -20 0C in 50 ,ul aliquots. Stock solutions of forskolin (50 mM), (± )Bay K 8644 (10 mM), (-)Bay K 8644 and PN 200-110 (10 mM) in dimethylsulphoxide (DMSO) were stored in aliquots at -20 0C. The final concentration of DMSO in the experimental solution did not exceed 041 %, and DMSO alone did not have any effects on ionic currents in the oocytes. 16

PHY 450

N. DASCAL AND OTHERS

472

Northern blot hybridization analysis The blot hybridization analysis was performed using standard methods (Maniatis et al. 1982). RNA was run on a formaldehyde 1 % agarose gel. The gel was stained with ethidium bromide to verify relative amounts of RNA, and then transferred to nitrocellulose and hybridized with 32P_ end-labelled cDHPR-02.

1. The composition of the solutions used in the study K+ NMDG+ Ca2+ Ba2+ Mg2+ TEA+ C1MS- 4-AP Solution 1 2 ND-96 1-8 103-6 142 2 40 Ba/MS 2 142 40 Ba/NMDG 60 142 2 2 40 40 20 Ba/NMDG/TEA 2 142 50 40 Ba/TEA 2 40 142 Ba/Na 60 2 142 40 20 Ba/Na/TEA 40 All concentrations are shown in mm. All solutions contained 5 mM-HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid), titrated to pH = 7-4-746 with NaOH in Na+containing solutions, and with Ba(OH)2 in solutions without Na+. TEA+, tetraethylammonium+; 4-AP, 4-aminopyridine; MS-, methanesulphonate-; NMDG+, N-methyl-D-glucamine. TABLE Na+ 96 60

Electrophysiological recordings The experiments were performed at room temperature (21-24 °C) using the solutions listed in Table 1. An oocyte was placed in a 1 ml bath, usually constantly perfused at a slow rate (1-5 ml/min) with the solution in which the recordings were performed. When the solutions were changed, the new solution was perfused at a higher rate (20-30 ml/min; the 'dead time' of the perfusion system was 5-10 s). The change in rate of flow solution either did not affect the Ba2' current (IBa) or sometimes caused a small increase in the current amplitude (up to 30%) that reversed in a few minutes after cessation of washing. Drug effects were tested after fast perfusion of about 10 ml of solution, as explained in the Results section. Cyclic AMP (2, 5 or 10 mm solution in water) was intracellularly pressure injected into the oocytes as described previously (Dascal et al. 1984); the injected volume did not exceed 10 nl (1 % of the oocyte volume). The electrophysiological recording system has been described elsewhere (Dascal et al. 1984; Dascal & Lotan, 1991 a). The cell was impaled with two conventional 3 M-KCl electrodes having resistances of 0-5-2 MQ. The bathing solution was connected to ground through a 3 M-KCl agar bridge. Two-electrode voltage clamp was performed using a Dagan 8500 amplifier (Dagan Corp., Minneapolis, MN, USA). Stimulation and data acquisition were done with an IBM XT computer supplied with a Techmar Labmaster ADC/DAC board, using the pCLAMP software (Axon Instruments, Burlingame, CA, USA). The membrane current was permanently monitored with a chart recorder. The currents evoked by the various experimental protocols were filtered at half the sampling frequency with an 8-pole Bessel filter and sampled at different rates (the Na+ current at 10 kHz, the Ba2+ currents at 100-1000 Hz, depending on the protocol) by the computer. In parallel, the membrane voltage and current were monitored with a dual-beam storage oscilloscope. When constructing I-V curves, the command voltage rather than the actually measured voltage was drawn at the abscissa. To avoid possible errors that could be introduced by this procedure, any cells in which, at the peak of the current, the measured values of voltage deviated by more than 2 mV from the command voltage were discarded. In most cells the deviation of measured voltage from the command voltage was within 0-5 mV.

Presentation of the data All mean values are shown with+ s.E.M.; the number of cells is shown in parentheses.

Ca2+ CHANNELS IN MUSCLE RNA -INJECTED OOCYTES

473

RESULTS

Expression of voltage-dependent Na+ channels Three or more days after the injection of total or poly(A)RNA extracted from skeletal muscle of 14- to 30-day-old rats, in normal Na+-containing solution (see A

B

~~+TTX

1

~-500 c

0.

-500

~~

~

~

-1000

~

~~~c-1000

Control

-1500

-1500. 7!'5

65 55 Time (ms)

45

C

Difference current

-Annn -.Luu, '

65 55 Time (ms)

45 D

to

1 00 -0-0o _ 100

250 0 c

-250 -500

-750 -1000

0

O/

x 80 (a E 60 40

/

co

0Gn

c \

0o

-80 -60 -40 -20 0 20 40 60 80 Vm (mV)

0

X

'o\ 0 0 0

o

20

c ___

75

n\ -100

-80

-60

-40

-20

Vm (mV)

Fig. 1. Voltage-dependent Na+ current in an SkM RNA-injected oocyte. Solution, ND 96. A, current evoked by a step from -100 to 0 mV in the absence (control) and in the presence of 100 nM-TTX. B, net INa obtained by subtraction of the upper trace from the lower trace shown in A. C, current-voltage relation of INa in the same cell. Net INa was obtained at each voltage as explained in B. D, steady-state inactivation curve of I,Na in the same cell. The current was evoked by depolarizing steps to -10 mV, following 50 ms prepulses to different voltages (shown at the abscissa).

Table 1; ND 96), the oocytes displayed a prominent, fast depolarization-activated current which ranged between 300 and 2500 nA in different oocytes and with different RNA batches (Fig. IA). This current was fully blocked by 100 nM-TTX (Fig. LA) or by replacing extracellular Na+ with N-methyl-D-glucamine (NMDG), but not by the removal of external Ca2+. Thus, the current reflected the activity of voltage-dependent Na+ channels (Hille, 1984). Na+ channels have also been expressed in the oocytes injected with RNA from brain, chick skeletal muscle, etc. (for reviews, see Dascal, 1987; Lester, 1988). Voltage-dependent TTX-sensitive Na+ currents have not been observed in native oocytes (that were not injected with RNA) used in this study, or in oocytes injected with 7-day rat heart RNA that expressed prominent Ca2+ channel currents (see below). Net Na+ current (INa) was obtained by digital subtraction of the current recorded in the presence of TTX (Fig. 1 B). The first 1P3-1P5 ms after the beginning of the voltage step were lost because of saturation of the recording system by the 16-2

474

N. DASCAL AND OTHERS

capacitative current (see Dascal & Lotan, 1991 a). However, the recorded 'Na appeared to faithfully reflect the usual features of this current such as a bell-shaped current-voltage relation which peaked at about -10 mV (Fig. 1 C) and a steadystate inactivation curve with half-inactivation around -60 mV and full inactivation A

E

100 0

Native (regular) 8

0

4-

80 60

O Native

(regular)

40 20

B

.

0I

L.- I...................................

1

10

100

1000

10

100

1000

ON°

2.5s

F

Native (variant) L-

4-i 0

a 0

4-

C

0

Muscle

1

G 100' 80

D 50 nA

0

s 0

N i2+

,c

Control

60

Peak

NON

40

Muscle

20

Heart

0

10

100

1000

[Ni2'] (#M)

Fig. 2. Voltage-dependent IBa and its inhibition by Ni2+. The currents were evoked by depolarizing steps from -100 to 0 mV in Ba/TEA solution (Table 1). Net IB was obtained by leak subtraction procedure, except B where currents recorded in the presence of 200 jim-Cd2+ were subtracted from control currents. A, C and D, IBa recorded in oocytes of one frog, either native (A), or injected with SkM RNA (C) or heart RNA (D), of 7-dayold rats. Control current, upper trace; current after the addition of 40 jM-Ni2+, lower trace. B, 'Ba in a native 'variant' oocyte. E-G, dose dependence of Ni2+ inhibition of IB. in oocytes of three different frogs: native regular (E), native variant (F), SkM RNA injected (G).

above -40 mV (Fig. 1D). INa served

as an

internal control in

many

of the

experiments described below; it was routinely recorded in Ca2+-free ND-96.

Ba2+ currents through Ca'+ channels in native and RNA-injected oocytes Ba2+ currents through Ca2+ channels (IBa) were recorded in Ca'+-free solutions containing 40 or 50 mm-Ba2 (Table 1). Na+ current was inhibited by replacing Na+ with NMDG or TEA (Table 1) or by adding 100 nM-TTX. The holding potential was

Ca2+ CHANNELS IN MUSCLE RNA-INJECTED OOCYTES

475

usually set to -80 mV. The depolarization-evoked inward current could not be carried by Cl-, because in many solutions (e.g. Ba/TEA, Ba/Na; Table 1), [CI-]. was 142 mm, and the predicted Cl- equilibrium potential was between -20 and -30 mV (Dascal et al. 1984). Thus, at voltages exceeding -20 mV, Cl- flow would be expected .to produce outward currents. In simple routine protocols, IBa was measured by 2-5 s test voltage steps to 0 or 20 mV following a 5 s prepulse to - 100 mV. The leak current was recorded with a step to -50 or -40 mV, and was scaled and subtracted digitally during the analysis session (Dascal et al. 1986; Lotan et al. 1989 b). In many cases, subtraction of currents evoked by the 'test' voltage steps in the presence of 200-500 ,tm-Cd2" was used instead of leak subtraction. The latter procedure gave better estimates of net IBa since it also eliminated the contribution of K+ currents. Native oocytes possess voltage-dependent Ca2+ channels, and depolarization in high-Ba21 solutions evokes a transient IBa, usually between -5 and -20 nA at 0 or 20 mV (Dascal et al. 1986; Umbach & Gundersen, 1987; Lory, Rassendren, Richard, Tiaho & Nargeot, 1990; Fig. 2A and Table 2). However, in some frogs the currents reached -30 nA, and in the oocytes of four frogs (out of the several tens of frogs tested), the native currents exceeded -30 nA (range -32 to -110 nA), and we termed these oocytes 'variant' (Fig. 2B and Table 2). A similar extreme variability of the endogenous muscarinic responses in the oocytes has been described (LupuMeiri, Shapira, Matus-Leibovitch & Oron, 1990). The reasons are not known but may be due to heterogeneity of the frog population, especially those caught in the wild (supplied from the African Xenopus Facility). About twenty extractions of RNA from skeletal muscle of 7-, 14-, 17-, 21- and 30day-old rats were performed. RNA of about 50% of the batches caused (3-5 days after RNA injection) the appearance of Ba2+ currents that were severalfold larger than in native oocytes (e.g. Fig. 2; compare C and A, both recorded in oocytes of the same frog). If RNA failed to induce the expression of IBa that was at least threefold larger than the IBa in native oocytes of the same frog, the experiment was discontinued. We also did not study the expressed Ca2+ channels in oocytes of 'variant' frogs (although we did observe a consistent increase in IB following SkM RNA injection in these cells). The Ba2+ currents expressed in oocytes injected with skeletal muscle RNA extracted from rats of different ages did not differ significantly in their amplitudes, shapes and other parameters (data not shown); thus, all data were pooled. The peak amplitude of IBa measured at 0 mV in SkM RNA-injected oocytes ranged between -40 and - 300 nA and was, on average, about fivefold larger than in regular native oocytes (Table 2). Even with 'good' RNA the expression of IB was highly variable among different donors, whereas INa was expressed faithfully and reproducibly. Thus, on one occasion, oocytes of two frogs were injected at the same day by the same person and after 4-5 days had a IN of -138+0-24 ,uA (frog 1, n = 6) and of - 1-28+0-19 ,tA (frog 2, n = 4), whereas IB. measured at + 20 mV was -20+ 8 nA in frog 1 (n = 6) and - 192 + 23 nA in frog 2 (n = 4). Native currents in oocytes of both frogs did not exceed -10 nA.

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Separation of two current components by Ni2+ The native Ba2+ current in 'regular' oocytes was transient and decayed to zero in less than 2 s (Fig. 2A). However, in 'variant' oocytes and in oocytes injected with skeletal muscle RNA, IBa appeared to display a biphasic decay (Fig. 2B and C). The slow component of the current was relatively sustained at 0 mV and was still TABLE 2. Amplitudes of Ba2+ currents in native and RNA-injected oocytes, and the effect of

100,um-Ni2+ Group

Amplitude (nA)

Inhibition by 100 gM-Ni2+ (%)

Native (regular) -110 + 1-2 (54) Native (variant) -62-9 + 4-9 (22) SkM RNA injected -56-7+4 8 (31) was evoked from to 0 mV. The entries are -100 IBa by steps given in parentheses.

94+1 (6) 73 + 7 (6) 59+5 (11) mean + S.E.M.; number of cells is

substantial 2-5 s after the beginning of the depolarizing pulse, resembling a slow current directed by rat heart RNA (Fig. 2D; see Dascal et al. 1986; Lotan et al. 1989b; Lory et al. 1990). A fast component of the current could be selectively dissected by inhibition with 40 gM-Ni2+, which had little (heart) or no (native 'variant', SkM) effect on the slow component (Fig. 2B-D). 'Regular' native IBa was reduced by 53 + 5 % at the peak (n = 8). The separation of the two components was further verified by studying the dose dependence of Ni2+ block in SkM RNA-injected oocytes, 'regular' native oocytes, and in two 'variant' native oocytes (Fig. 2, E-G). The concentrations of Ni2+ that produced 50% block of the total peak IBa (IC50) did not differ greatly in 'regular' native and SkM RNA-injected cells: they were 29 + 7 (n = 4) and 45 + 8 (n = 8) gm, respectively. However, two characteristics were noteworthy. (i) There was no detectable IBa at 500 m-MNi2+ in regular native oocytes, whereas in RNA-injected oocytes a prominent Ba2+ current still remained. The sensitivity of IB. to Ni2+ is illustrated by the effect of 100 ,m-Ni2+ (Table 2): at this concentration, 95 % of the peak IB. in regular native oocytes was inhibited, whereas peak IBa in SkM RNA-injected oocytes was inhibited only by 59%. The sensitivity of IBa in the 'variant' native oocytes to Ni2+ was intermediate (inhibition by 73% in 100 gMNi2+). (ii) The current measured in native 'variant' and in SkM RNA-injected oocytes at the end of the 2-5 s pulse was much less sensitive to Ni2+ block than at the peak, being only slightly (usually by 10-20°%) reduced at 100 or 200 /M-Ni2+ (Fig. 2F and C). Thus, the curves representing the dose dependence of the Ni2+ block of IBa measured at this time point reflected mostly the block of the slow component. These data led us to suggest that IB. in 'variant' native oocytes and in SkM RNAinjected oocytes consists of at least two components: a fast, Ni2+-sensitive current, almost fully inhibited by 100 gtM-Ni2+, and a slow, Ni2+-resistant IB., only marginally (5-15 %) inhibited by 100 gM-Ni2+; the Ni2+-resistant IBa is absent or is very small in 'regular' native oocytes. In the following, 100 (rarely 200) gM-Ni2+ was used to inhibit the faster component. Obviously, separation by Ni2+ was not perfect. We

Ca2` CHANNELS IN MUSCLE RNA-INJECTED OOCYTES

477

estimated that the currents separated by Ni2+ were 'contaminated' by 10-15% by each other. None the less, such separation was helpful in revealing differences between Ni2+-sensitive and Ni2+-resistant currents (see below). Kinetics and voltage dependence of Ni2+sensitive and Ni2 -resistant IBa i S RNAinjected oocytes In the study of voltage dependence and kinetics of 'Ba' the currents were recorded in control (blocker-free) solution, then in the presence of 100 (rarely 200) /,M-Ni2+, to

A

B o

Ni2 sensitive

0

-50

-50

-100

005 56-8+1-7 61-1+09 -4-3+1t6 Vr(mV) n.d.* 3 0+005 2-3+0 3 Gmax (11S) < 0 01 -8-9+1 1 5-6+0 2 -14-5+1 2 Va (mV) > 01 8-3+0-5 94+07 -1-1+0-6 Ka(mV) Inactivation Ni2+ resistant P Ni2+ sensitive Difference V (mV) < 0 01 -40-9+3 0 -12-2+3 0 -28-7 +3-3 > 005 2-1+0-8 13-6+0-8 11P5+0-3 Ki(mV) and inactivation curves of Ni2+-sensitive and The parameters of Boltzmann equations for I-V Ni2+-resistant currents were obtained in four cells. The equations used in the fits are given in the legend to Fig. 4. The correlation coefficients of the fits of the various curves in all cells were better than 0-98. Entries are mean+S.E.M. P was calculated using Student's paired two-sided t test. * Difference for the Gm.x parameter is meaningless and has not been determined (n.d.). The ratio of GmaX values of the Ni2+-sensitive and Ni2+-resistant IB., which may give some estimate of the relative contribution of each channel type to the total current, was 0-83+0-13 in these cells.

The voltage-dependent inactivation of total and Ni2+-resistant 'Ba is illustrated in Fig. 4C and D. In most cells, the steady-state inactivation curve had a small but consistent 'hump' suggesting the existence of two populations of channels (Fig. 4 G). Upon separation by Ni2+, the steady-state inactivation curves of Ni2+-sensitive and Ni2+-resistant currents displayed clear differences (Fig. 4G): although the two curves were practically parallel and had similar slope factors (Ki), the halfinactivation voltage (Vi) of Ni2+-sensitive IBa was about 30 mV more negative than that of Ni2+-resistant IBa (Table 3). Thus, the faster, Ni2+-sensitive IBa inactivated at much more negative voltages than the Ni2+-resistant IBa

Effects of a Ca2+ channel agonist and blocker on IBa In SkM, the classical L-channel has a distinct pharmacology, being highly sensitive to DHP blockers and enhancers ('agonists') and other organic blockers such as phenylalkylamines (e.g. verapamil) and benzothiazepines (e.g. diltiazem) (for review, see Glossmann & Striessnig, 1990). In initial experiments, the racemic mixture, (± )Bay K 8644, appeared to enhance the IBa in some SkM RNA-injected oocytes by up to 30% (cf. Lotan et al. 1989 a). However, careful examination of this effect suggested it might result from an increase of IBa due to washing (see Methods). Therefore, further tests of the various drugs have been performed under thoroughly controlled conditions in which IBa was recorded after extensive washing with the same amount (usually 10 ml) of either control or drug-containing solution.

Ca2+ CHANNELS IN MUSCLE RNA -INJECTED OOCYTES C

B

A

481

Muscle

Native (variant)

Heart

Control Control, Bay K

Bay K 100 nA

is F

E

D

c

Control, PN 200-1

Native

Muscle

Muscle

a, b

b

Fig. 5. Effects of organic blockers and an agonist on IB.. A, effect of 1 ,M-( ± )Bay K 8644 in an oocyte injected with heart RNA. Solution, Ba/NMDG/TEA, in the presence of 40 sMm-Ni2+. B, effect of 1,uM-(-)Bay K 8644 in a native variant oocyte. Solution, Ba/NMDG. C, effect of 1 gM-(-)Bay K 8644 in an oocyte injected with SkM RNA. Solution, Ba/NMDG. D, effect of 1 gM-( + )PN 200-110 in an SkM RNA-injected oocyte. Solution, Ba/NMDG/TEA. E, effect of 100 ,UM (b) and 500 /M (e) diltiazem on an SkMinjected oocyte. Trace a shows I,. before the addition of diltiazem. Solution, Ba/NMDG/TEA. F, same as E, but in a native regular oocyte. Same frog and solution as in E. TABLE 4. The effect of (± )Bay K 8644 and (-)Bay K 8644 on 'Ba 1-2 ,UM-(-)Bay K 1-2 ,uM-(± )Bay K 10-100 nM-(-)Bay K 98+4 (3) 87±6 (2) 98+2 (6) Native 95+4 (5) 101+2 (5) 107+3 (7) SkM RNA injected The entries represent the mean (+ S.E.M.) amplitudes of I,. expressed as percentage of control, i.e. of IBa measured in the same cell before administration of Bay K 8644. The number of oocytes is given in parentheses.

(± )Bay K 8644 had no effect on IBa in native ('regular' and 'variant'; Fig. 5B) and in SkM RNA-injected oocytes (see Table 4 for a summary). The (-)enantiomer of Bay K 8644, reported to be a pure agonist devoid of antagonistic activity (see Glossmann & Striessnig, 1990), caused no enhancement of IBa in SkM RNA-injected oocytes (Fig. 5C; Table 4). The absence of a substantial effect of Bay K 8644 was in clear contrast with a 100-200% enhancement, by the same concentrations of this drug, of IBa expressed in oocytes injected with heart RNA (Lotan et al. 1989b; Lory et al. 1990; Fig. 5A). Two potent DHP Ca2+ channel blockers, nifedipine (10 /UM, three cells) and (+ )PN 200-110 (1-10 /UM, two cells), did not cause any detectable block Of IBa in SkM RNAinjected oocytes kept at a holding potential of -80 mV (Fig. 5D). Activation and steady-state inactivation curves of IBa were not affected by (+ )PN 200-100 (two

482

N. DASCAL AND OTHERS

cells, not shown). Testing the effect of DHP antagonists at a depolarized holding potential (-40 mV) turned out to be impossible because of a slow inactivation process that led to a complete disappearance Of IBa in the course of a few minutes (not shown). Verapamil did not affect IBa in SkM RNA-injected oocytes at 50 JtM (two cells), and reduced the current by 5-20 % at 100 /IM (two cells). Repetitive stimulation at a frequency of 0 33 Hz caused a further reduction of IBa (to 72-75 % of control) in the presence of 100 /uM-verapamil, suggesting that there might be a use-dependent effect of the drug; however, repetitive stimulation of the oocyte at similar frequencies in the absence of any blocker similarly reduced the current (not shown), possibly due to incomplete recovery from the slow inactivation process. Diltiazem significantly reduced IBa in SkM RNA-injected and in native oocytes (Fig. 5E and F). Diltiazem at 100 ,tM reduced IBa by 26 + 4 % (n = 8) in SkM RNAinjected oocytes, and by 13+3 % (n = 3) in native oocytes; 500,M reduced the currents by 52+3 (n = 3) and by 50 + 6 % (n = 3), respectively. In SkM RNAinjected oocytes, diltiazem appeared to reduce both Ni2'-sensitive and Ni2+-resistant IBa (two cells; not shown). Note that concentrations of diltiazem and verapamil needed to block IBa in SkM RNA-injected and native oocytes were at least two orders of magnitude higher than in SkM; at such concentrations, these agents can block T- and N-type Ca2+ channels (see Glossmann & Striessnig, 1990) and cannot be considered specific for the Lchannel.

Cyclic AMP potentiates IBa in native and SkM RNA -injected oocytes Figure 6 shows that IBa was enhanced by intracellular pressure injection of 4-20 pmol cyclic AMP in SkM RNA-injected oocytes (by 82 + 17 %, n = 6; Fig. 6A) as well as in native oocytes (by 94 + 19 %, n = 3; Fig. 6B). The onset of the cyclic AMP effect was 1-2 min after the injection; it reached a maximum after 7-10 min, and faded over the next 10-15 min (Fig. 6C). Additional injections of the same or higher doses of cyclic AMP again elevated IBa (Fig. 6C). Forskolin (an adenylate cyclase activator; 20-50 /tM) also potentiated IBa in SkM RNA-injected oocytes, but less potently than cyclic AMP injection (27 + 6 % increase, n = 4). The IB. enhanced by cyclic AMP or by forskolin was not sensitive to (-)Bay K 8644 (five cells), suggesting that the effect of cyclic AMP was not due to the appearance of a previously 'dormant' population of L-channels. Selective destruction of the RNA coding for the ac1-subunit of S/cM Ca2+ channel does not suppress the expression of Ca21 channels in SkM RNA -injected oocytes Since the sequence of the cDNA coding for the main, channel-forming (acJ) subunit of the L-type Ca21 channel is known (Tanabe, Takeshima, Mikami, Flockerzi, Takahashi, Kangawa, Kojima, Matsuo, Hirose & Numa, 1987; Ellis, Williams, Ways, Brenner, Sharp, Leung, Campbell, McKenna, Hui, Schwartz & Harpold, 1988), it is possible to probe the molecular identity of the Ca2+ channel(s) expressed in oocytes injected with skeletal muscle RNA, using the hybrid arrest method (see Lotan et al. 1989 b, and references therein). The procedure is as follows: heterologous RNA from the tissue under study is hybridized with a 50-80 bases DNA

Ca2+ CHANNELS IN MUSCLE RNA-INJECTED OOCYTES 483 oligonucleotide complementary to the RNA corresponding to the cloned cDNA, and the mixture is injected into the oocyte. There, the RNA moiety of the RNA-oligonucleotide hybrid is attacked and degraded by an RNAaseH-like activity. Thus, expression of the ion channel encoded by the RNA corresponding to the cloned A

B

Muscle

Native

_~~Control

*,