Exp. Eye Res. (1992) 55, 861-868
Potassium
Currents KIM
Department
COOPER*,
from
Isolated
MITCHELL
Frog Lens Epithelial
WATSKY”AND
JAMES
Cells
RAE”
of Zoology, Arizona State University, Tempe, AZ, and aDepartments of Physiology Biophysics and Ophthalmology, Mayo Foundation, Rochester, MN, U.S.A. (Received Bethesda 2 April
and
7991 and accepted in revised form 27 April 1992)
Using the perforated patch version of whole-cell recording, we have measured currents from isolated frog lens epithelial cells. Three types of currents were seen. A time-independent outwardly rectifying. potassium current was identified that sets the resting voltage. This potassium current differs significantly from any of the potassium currents recorded with the whole-cell technique in mammalian lens epithelial cells. In addition to the potassium current, the two other currents present were both outwardly rectifying : one was time-independent while the other showed distinct activation. Key words: frog lens epithelium : K current ; resting voltage : ion transport ; voltage clamp ; patch clamp.
1. Introduction
Potassium conductance is involved in determining the cellular ionic steady-state necessary for cells to control their volume (Guggino, 1989). The precise control of cell volume in the lens is required to maintain the very small extracellular space(approximately 2 %) essential for maximum transparency (Rae, 1979). The potassium conductance is also an essential element of the hypothetical lens internal circulation system proposed by Mathias (1985). According to the Mathias model, the potassium conductance is localized in the lens surface while the sodium and chloride conductances are restricted to the lens interior. This spatial heterogeneiti of conductance sets up a standing current that drives a standing water flow. The water flow acts as an internal circulation for the lens. The lens potassium conductance is also important in controlling protein synthesis. cc-Crystallip synthesis is known to depend on the cellular sodium/potassium concentration ratio (Shinohara and Piatigorsky, 1977). Studies on whole lenses have shown that the potassium conductance is indeed located predominantly in the outer layers of the lens, including the epithelium (Duncan, 1969; Mathias et al., 1985). Recent work using the whole-cell recording technique on isolated lens epithelial cells has begun to reveal a fairly simple story regarding the molecular identity of the currents underlying this potassium conductance. In mammalian lens epithelial cells, there are three main potassium currents, an outwardly rectifying current (Cooper et al., 1990), an inwardly rectifying current (Cooper,Rae and Dewey, 199 1) and a calcium activated potassium current (Rae and Cooper, 1990 ; Copper et al., 1990). The role of the calcium activated current has not yet been determined, but the other * For correspondence at: Department of Zoology, Arizona State University, Tempe. AZ 85287-l 501, U.S.A. 00144835/92/120861+08
$08.00/O
two currents are active under physiological conditions and are likely to be important in setting the mammalian lens resting voltage. Different mammalian specieshave differing relative amounts of the inwardly and outwardly rectifying currents. Historically, the most studied lens, from an electrophysiological standpoint, is that of amphibians. Even though it has been well established from macroscopic measurements that this preparation has a large surface potassium conductance (Mathias et al., 198 S), single channel recording has consistently failed to identify a potassium channel in sufficient density to explain the macroscopic results (Jacob, 1984; Rae, 1985). The motivation for this study was to see if the potassium conductance of amphibian lens could be demonstrated using whole-cell voltage clamp recording, and to seeif the potassium conductance is similar to that in the mammalian preparations. Our major finding is that the potassium conductance is easily demonstrated and that it is different from that seen in mammalian species. In addition to this potassium current, we also noted the existence of two other currents. 2. Materials and Methods Cell Dissociation Rana pipiens were housed at room temperature and fed daily. The eyes were removed and the lenses dissectedfree from the globe using standard procedures (Cooper, Rae and Gates, 1989). The lens capsule was peeled from the posterior surface and pinned to a Sylgard disc. The fiber cell masswas then removed and the remaining epithelial sheet-capsule preparation was cleaned of any adhering fiber cell debris. The disc holding the epithelium was transferred to one well of a multi-well plate containing 0.08% trypsin (Sigma type III) and 2 mM EGTA in normal Ringer’s solution. 0 1992 Academic Press Limited
K. COOPER
862
ET AL.
TABLE I Solution composition
2.5 104 107 22.5
Normal Ringer Pipette Solution 107K 22K
MeS* Na ___-. 0 104.5
Cl
K
Solution
114 10
100
10
2.7 12.7
111.3
5 89.5
101.3
Ca
EGTA
SO,
Mg
2
0 2
0 0 1.5 1.5
0 1.5 1.5
0 1
0
1
0
1.5
* Methane sulfonate.
(A)
a a 8
L5 msec
~_____.
80 70
0 60 50a ,a ; E 3
0
40
0
30-
0 0
zoI 0
IOO0
Ooo -IO
-120
_!L--80
_ u
0
0
-40
I 40
0 Voltage
I 80
I I20
i
hV1
FIG. 1. A, Whole-cell currents in responseto a step voltage clamp protocol. The cells were bathed in normal Ringer’s solution, The voltage pulses were 40 msec in duration and went from - 140 mV to + 140 mV in 20 mV increments. The cells were held at - 60 mV between pulses. This current is the average of five repetitions of the protocol. In this and all subsequent figures, capacitance transients have been edited. B. Current-voltage relationship from (A), measured at the end of the voltage pulse. The cells remained in this solution for 30 min. At the end of this period, the disc was transferred to a well containing normal Ringer’s solution. The cells were gently triturated off the capsule using a firepolished Pasteur pipette. The solution containing the isolated cells was then transferred to a conical tube until use.
Whole-cell Recording A drop of solution containing isolated cells was placed in a custom built chamber (volume about 300 ~1) attached to the stage of an inverted Nikon Diaphot microscope. Cells typically attached within
FROG
LENS
POTASSIUM
CURRENT
5 min to a piece of microscope slide glass placed in the bottom of the chamber within 5 min. Whole-cell recording was done using standard techniques (Rae and Levis, 1984). Electrodes were made of Corning 7052 glass. After fire polishing, the electrodes had resistances of about 4.5 MQ. Recordings were made using a modified Axopatch-1B patch clamp (Axon Instruments, Foster City, CA). The data were digitized using a TL-1 A/D-D/A interface (Axon Instruments) into a modified IBM-AT computer. Data were acquired and analyzed using the pCLAMP software package (Axon Instruments). Recordings were made with a bandwidth 5 kHz and sampled every 100 psec. The whole cell time constant determined by the electrode accessresistance and the cell capacitance was typically about 160 psec. Capacitance transients were cancelled electronically. Any residual transient was removed using Drawperfect (WordPerfect Corp., Orem, UT). All experiments were performed at room temperature. The isolated cells had a mean diameter of 17.3 f0.4 pm (mean? s.E.M.; n = 47) and a mean capacitance of 11-4 + O-5 picoFarads (n = 47). Recordings were made using a modified version of the perforated patch recording technique (Rae et al., 1991). In brief, this technique uses the pore forming ant.ibiotic amphotericin-B to produce a low access resistance pathway from the recording pipette into the cell interior. The amphotericin-B is added to the pipette filling solution in a concentration of 240 ,ug ml-’ (about 2.5 x 10Y4mM). The tip of the electrode is filled to approximately 500 i&m with amphotericin-B free intracellular solution. This was necessary because amphotericin-B interferes with seal formation. Amphotericin-B takes about 10 min to diffuse to the tip and partition into the patch membrane in sufficient quantity to allow recording. The advantage of this technique over traditional whole-cell recording is that the amphotericin-B pore is only permeable to small monovalent ions. Thus, the concentrations of control molecules, like CAMP and calcium, are not altered during recording. The final accessresistance was usually about 1.5MR. Because of the low access resistance and small currents (approximately 100 PA), series resistance compensation was unnecessary (maximum voltage error under 5 mV).
863
as needed with the membrane impermeant anion methane sulfonate. The pipette solution was designed to mimic the internal composition of frog lens epithelial cells. Normal cellular impermeant anions were balanced by the membrane impermeant anion, methane sulfonate and internal calcium was buffered to a low level using EGTA. However, given that amphotericin-B is divalent impermeant. the exact value of the pipette calcium is unimportant. 3. Results Potassium Current
Figure 1(A) shows the responseof frog lens epithelial cells to a series of voltage clamp pulses. The pulses IOOr
Voltoge
(mV)
FIG. 2. Potassiumselectivity of whole-cell current. Cur-
rent-voltage relationships from a cell in three different external potassiumconcentrations.(0) 2.5 K: (V) 22.5 K:
Solutions
The composition of all solutions is given in Table I. All solutions were buffered with 5 mM Hepes. The external solutions were buffered to a pH of 7.35, and the internal solution to 7.00. All solutions had an osmolality of 22 5 mosm kg-‘. The 107K and 22K solutions are modified Ringer solutions used to test the selectivity of the whole-cell current. They were designed to have a nearly constant KC1 product to minimize cell volume changes. To maintain the constant KC1product, Cl was replaced
FIG. 3. Barium block: (0) control current-voltage relationship : (0) current-voltage relationship after addition of 5 mM barium to the bath: (0) differences between control and barium current-voltage relationships.
K. COOPER
864
ET AL.
(A)
20 msec
-__
IO00 (El 800
c
Voltage
(rnv)
4. Time-dependentcurrent. A, Current in responseto a family of voltagestepsof 100 msecduration. Pulseswent from - 120 mV to +20 mV increments.Five repetitionswere averaged.B, Current at the end of the lOO-msecpulsein (A) plotted againstpulsevoltage. FIG.
were of 40 msec duration and spanned a voltage range from - 140 to 140 mV in 20 mV increments. In general, five repetitions were averaged to produce the data. The cells were held at - 60 mV during the interpulse period. Figure l(B) shows the steady-state current-voltage relationship from Fig. l(A). The current-voltage relationship is roughly exponential, with outward currents being larger than inward currents from the same driving force. This behavior was representative of that seen in 40 of the 47 cells studied. There was no significant time dependenceto the current. The cell’s resting voltage was taken to be the zero current point on the current-voltage curve. The average resting voltage of these cells was - 56 f 4 mV (n = 47). The cell input resistance was determined from the slope of the current-voltage relationship in the vicinity of the resting voltage. The average value was 4.9 + O-6 GQ (n = 47).
To demonstrate the potassium selectivity of the resting potential, the bath solution was exchanged for a series of solutions containing varying potassium concentrations. Figure 2 shows the resulting currentvoltage relationships for external potassium concentrations of 2.5, 22.5 and 107 mM. The resting voltage shifted in the direction expected for a potassium selective membrane, and the inward current increased with increasing external potassium concentration as expected. Similar behavior was seen in five cells. The residual reversal potential in the high potassium solution represents an artifactual offset voltage. Since internal and external solutions are nearly identical, it seems unlikely that this offset was due to a residual membrane current. The possibility that this offset was due to the sodium pump was examined by adding 0.1 mM ouabain to the bath. No consistent change in the offset was seen.
FROG
LENS
POTASSIUM
CURRENT
865
I
5 msec
I
a a 0" L-
-
5 msec
FIG. 5. Current activation. A, Control current in response to 50 msec pulses from - 100 mV to + 140 mV. B, Current from same cell 15 min later. Voltage pulses go from - 140 mV to + 140 mV.
In an attempt to isolate the potassium current, the potassium channel blockers barium and tetraethylammonium (TEA) were used. TEA was found to be an ineffective blocker at concentrations up to 10 mM (data not shown). Figure 3 shows the control steady-state current-voltage relationship (open circles) and the current-voltage relationship after block by 5 mM barium (filled circles). The resting voltage shifts from - 60 mV to 0 mV in the presence of barium. The difference between these two current-voltage relationships is the potassium current of interest (triangles). The difference current reverses at a voltage very near the calcuIated potassium reversal potential (- 95 mV), and is thus a potassium current. The current is outwardly rectifying and declines at voltages above 50 mV. The currents before and after barium addition had no time dependence.Similar behavior was seenin nine other cells. Other Currents
In addition to the potassium current described
above, a time-dependent current was seen in seven of the 47 cells investigated. Figure 4(A) shows this current. It exhibits slow (about 100 msec time constant) activation kinetics. Figure 4(B) shows the steady-state current-voltage relationship from Fig. 4(A). The current shows outward rectification, becoming significant only at voltages above 0 mV. The fact that the current reversesat 0 mV could imply that this is a nonselective current. Another possibility is that the current is the sum of a non-selective leak (e.g. current through the seal) and a voltage-dependent current that does not activate until around 40 mV. The selectivity of such a current can not be determined from this experiment. In four of the ceils, the current increased dramatically during recording as shown in Fig. 5. This often coincided with a noticeable increase in cell diameter. When barium was applied, the activated current could be separated into two components. Figure 6(A) shows the current remaining after barium addition. The current-voltage relationship is presented in Fig. 6(B). The current reverses at 0 mV and has the
K. COOPER
866
ET AL.
2500 z 2002 5 150L =
0
IOO0 50-
0 C’ O0
O(W -50 -50
1 II -120
I -80
I -40
I 0 Voltage
I 40
I 80
I I 120
(mV)
FIG.6. A, Current from cell in Fig. 5 after addition of 5 mM barium. B, Current-voltagerelationship after barium. samegeneral shape as the current-voltage relationship shown in Fig. 4. Despite these similarities, the current is lacking the large tail seen in Fig. 4(A). This may represent block of the inward current by barium. It is also noteworthy that the tie course of activation is much faster than in Fig. 4(A). The difference between Fig. 5(B) and 6(A) is the current blocked by barium [Fig. 7(A)]. The steady-state current-voltage relationship is shown in Fig. 7(B). The negative reversal potential, outward rectification, and decline above 50 mV are all similar to the potassium current identified in Figs 1-3. Although the steady-state current is similar to the control potassium current, it has an early transient decay that is not seen in the control case. This may represent yet another current. 4. Discussion The properties of the cells studied here are comparable to those reported in previous work on isolated frog lens epithelial cells (Rae, 1985 ; Cooper et al., 1989). In particular, the mean resting voltage of - 56 mV agrees with that reported in several other
studies (Jacob,1984; Cooper et al.. 1986). However, it should be noted that there are several potential problems associatedwith the measurement of resting voltage using the perforated patch technique. First, the measured resting voltage (V,,,,) is only an approximation of the true resting voltage (V,). This is becausethe parallel combination of the seal resistance (R,) and the cell input resistance (R,) act as a current divider, resulting in : Vmeas= V,*R,/(R, + Rm)
It is difficult to accurately measure, I?, using the amphotericin-B technique, because the seal is in parallel with the accessresistance through the patch. The amphotericin-B partitions into the patch membrane so quickly that the access resistance is never high enough relative to R, to allow an accurate measurement of R,. A reasonable estimate of the seal resistance is about 20 GQ. Given that the input resistance of these cells is around 5 GQ, the measured resting voltage could be too low by 20% or more. In addition to this error, there are errors due to junction potentials and a Donnan potential across the ampho-
FROG
LENS
POTASSIUM
CURRENT
867
(A)
a a g- l-
5 msec
-‘“OF I
-2OO’-,
I 20
I
-80
I
-40
Voltage
I
0
I
40
I
80
I
120
I
;
(mV)
FIG. 7. A, Difference between currents in Figs S(B) and 6(A). B, Current-voltage relationship of difference current.
tericin-B (Horn and Marty, 1988). Also, whenever whole-cell recording is done, the internal concentrations of potassium, sodium, and chloride are altered, hence changing the diffusion potentials for these ions. All of these errors are hard to quantitate and thus make an absolute measurement of resting voltage difficult. The good agreement with previously published results on these cells gives us some confidence that these errors are not too extreme. The major result of this paper is the identification of the potassium current responsible for the resting voltage in frog lens epithelium. Figure 3 demonstrates the large shift in resting voltage upon addition of barium to the bathing solution. The current blocked by barium is unlike any of the potassium currents seen using whole-cell recording in mammalian lens epithelia. It is outwardly rectifying, like one of the potassium currents seen in mammalian lens, but shows none of
the characteristic time dependence (Cooper et al., 1990). The frog lens potassium current reported here is not blocked by TEA but is blocked by barium. The outwardly rectifying potassium current in human lens epithelia responds to these blockers in exactly the opposite way (Cooper et al., 1990). The rectification of this current is similar to the Goldman constant field rectification expected from the diiering concentrations of potassium on the two sides of the membrane. This can be seen in Fig. 2 where the inward current increases as external potassium is increased. The rectification appears instantaneously. This implies one of two things: either the channel open probability is voltage dependent but the gating is too fast for the recording apparatus to seethe opening process; or the channel might have a voltage independent open probability and the rectification could be an open channel property.
K. COOPER
Another interesting property of this current is the noticeable decrease in current above 50 mV. This could arise from several mechanisms. It could be due
to a voltage dependent inactivation process. If this is the case, the rate of this process is again faster than the recording apparatus can detect. It could also be due to voltage-dependent block by an internal cation such as magnesium, as is true in some types of inward
rectifiers (Nonner and Adams, 1990). Finally, it may be an artefact due to barium activation of some outwardly rectifying current. The single-channel basis of this potassium current is unknown and it is certainly possible that more than one type of potassium channel could be involved. Single channel recording from frog lens epithelia has shown the existence of a 40 pS potassium channel (Rae, Levis and Eisenberg, 1988), but its density seems too low to explain the current reported here. If this channel were open 20 % of the time, approximately 50 such channels would be necessary to explain the magnitude of the whole cell current. Given that the cells have an area of about 1000 ym2, and a patch of membrane in a single-channel experiment is on the order of 10 pm”, one would expect to seesuch a singlechannel in roughly every other patch. However, the reported density was one potassium channel in about
1000 patches. Such discrepancies between singlechannel and macroscopic current recording are not uncommon (Cooper et al., 199 1).
Reports of macroscopic measurements of potassium conductance in amphibian lens show somesimilarities to the data reported here. The exponential shape of the current and the barium sensitivity are similar to that
reported from whole frog lens (Delamere, Duncan and Paterson, 1980). Also, recent experiments in toad Iens using short circuit current measurements have identified a pH sensitive potassium conductance that may
be involved in volume regulation (Alvarez, Wolosin and Candia, 1991). This current is sensitive to barium.
In addition to the potassium current, two other currents were seen: one was time-independent and the other was a time-dependent outward rectifier. The physiological role of these currents is unclear, but if
either of them is a non-selective current in might explain why the lens resting voltage is depolarized relative to the potassium reversal potential. The barium sensitive component of the activated current has many properties similar to the potassium current, In summary,
whole-cell
recording
has allowed
identification of the major current types in isolated frog lens epithelial celIs. These include: an outwardly rectifying
time-independent
potassium current,
and
two other outwardly rectifying currents. These currents might be sufficient to explain the ionic basis of the resting voltage in the frog lens. The potassium current is distinct from any of the whole-cell recorded
potassium currents from mammalian lens epithelia.
ET AL.
Acknowledgements The authors would like to acknowledge Erika Wohlfiel for expert technical assistance in preparing this manuscript. This work was supported by NIH grants EYO3282. EY06005, EY09636 and an unrestricted award from Research to Prevent Blindness. References Alvarez, L. J., Wolosin, J. M. and Candia, 0. A. (1991). Contribution for a pH-sensitive and tonicity-sensitive K” conductance to toad translens short-circuit current. Exp. Eye Res. 52. 283-92.
Cooper, K.. Gates, P., Rae, J. L. and Dewey. J. (1990). Blectrophysiology of cultured human lens epithelial cells. 1. Membr. Biol. 117, 285-98. Cooper, K., Rae, J. L. and Dewey. J. (1991). Inwardly rectifying potassium current in mammaliam lens epithelial cells. Am. J. Physiol. 261, Cl 15x123. Cooper, K., Rae, J. L. and Gates, P. (1989). Membrane and junctional properties of dissociated frog lens epithelial cells. 1. Membr. BioI. 111, 2 15-2 7. Cooper, K., Tang, J. M., Rae, J. L. and Eisenberg,R. S. (1986). A cation channel in frog lens epithelia responsive to pressure and calcium. 1. Membr. BioZ.93, 25949. Delamere, N. A., Duncan, G. and Paterson, C. A. (1980). Characteristics of voltage-dependent conductance in the membranes of a non-excitable tissue: the amphibian lens. 1. Physiol. 308, 49-59. Duncan, G. (1969). The site of the ion restricting membranes in the toad lens. Exp. Eye Res. 8, 406-12. Guggino, W. B. (1989). Potassium and cell volume. In The Regulation of Potassium Balance (Eds Seldin, D. W. Giebisch, G.). Pp. 121-137. Raven Press, Ltd.: New York, U.S.A. Horn, R. and Marty, A. (1988). Muscarinic activation of ionic currents measured by a new whole-cell recording method. 1. Gen. Physiol. 92, 145-59. Hughes, B. A. and Steinberg, R. H. (1990). Voltagedependent currents in isolated cells of the frog retinal pigment epithelium. I. Physiol. 248, 273-97. Jacob,T. J. C. (1984). Three types of channel activity in frog lens epithelial cells. Exp. Eye Res. 38. 657-60. Mathias, R: T. (1985). Steady-statevoltages, ion fluxes, and volume regulation in syncytial tissues. Biophys. J. 48, 435-48. Mathias, R. T., Rae. J. L.. Ebihara, L. and McCarthy, R. T. (198 5). The localization of transport properties in the frog lens. Biophys. J. 48, 423-34. Rae, J. L. (1979). The electrophysiology of the crystalline lens. Curr. Topics Eye Res. 1, 3 7-90. Rae, J. L. (1985). The application of patch clamp methods of ocular epithelia. Curr. Eye Res. 4. 409-20. Rae, J. L. and Cooper. K. (1990). New techniques for the study of lens electrophysiology. Exp. Eye Res. 50, 603-6. Rae, J., Cooper, K., Gates, P. and Watsky, W. (1991). Low access resistance perforated patch recordings using amphotericin B. 1. Neurosci. Methods 37, 15-26. Rae, J. L. and Levis, R. A. (1984). Patch voltage clamp on lens epithelial cells: theory and practice. MoI. Physiol. 6, 115-62.
Shinohara, T. and Piatigorsky, J. (1977). Regulation of protein synthesis, intracellular electrolytes and cataract formation in vitro. Nature 270, 406-11.
Potassium
Currents KIM
Department
COOPER*,
from
Isolated
MITCHELL
Frog Lens Epithelial
WATSKY”AND
JAMES
Cells
RAE”
of Zoology, Arizona State University, Tempe, AZ, and aDepartments of Physiology Biophysics and Ophthalmology, Mayo Foundation, Rochester, MN, U.S.A. (Received Bethesda 2 April
and
7991 and accepted in revised form 27 April 1992)
Using the perforated patch version of whole-cell recording, we have measured currents from isolated frog lens epithelial cells. Three types of currents were seen. A time-independent outwardly rectifying. potassium current was identified that sets the resting voltage. This potassium current differs significantly from any of the potassium currents recorded with the whole-cell technique in mammalian lens epithelial cells. In addition to the potassium current, the two other currents present were both outwardly rectifying : one was time-independent while the other showed distinct activation. Key words: frog lens epithelium : K current ; resting voltage : ion transport ; voltage clamp ; patch clamp.
1. Introduction
Potassium conductance is involved in determining the cellular ionic steady-state necessary for cells to control their volume (Guggino, 1989). The precise control of cell volume in the lens is required to maintain the very small extracellular space(approximately 2 %) essential for maximum transparency (Rae, 1979). The potassium conductance is also an essential element of the hypothetical lens internal circulation system proposed by Mathias (1985). According to the Mathias model, the potassium conductance is localized in the lens surface while the sodium and chloride conductances are restricted to the lens interior. This spatial heterogeneiti of conductance sets up a standing current that drives a standing water flow. The water flow acts as an internal circulation for the lens. The lens potassium conductance is also important in controlling protein synthesis. cc-Crystallip synthesis is known to depend on the cellular sodium/potassium concentration ratio (Shinohara and Piatigorsky, 1977). Studies on whole lenses have shown that the potassium conductance is indeed located predominantly in the outer layers of the lens, including the epithelium (Duncan, 1969; Mathias et al., 1985). Recent work using the whole-cell recording technique on isolated lens epithelial cells has begun to reveal a fairly simple story regarding the molecular identity of the currents underlying this potassium conductance. In mammalian lens epithelial cells, there are three main potassium currents, an outwardly rectifying current (Cooper et al., 1990), an inwardly rectifying current (Cooper,Rae and Dewey, 199 1) and a calcium activated potassium current (Rae and Cooper, 1990 ; Copper et al., 1990). The role of the calcium activated current has not yet been determined, but the other * For correspondence at: Department of Zoology, Arizona State University, Tempe. AZ 85287-l 501, U.S.A. 00144835/92/120861+08
$08.00/O
two currents are active under physiological conditions and are likely to be important in setting the mammalian lens resting voltage. Different mammalian specieshave differing relative amounts of the inwardly and outwardly rectifying currents. Historically, the most studied lens, from an electrophysiological standpoint, is that of amphibians. Even though it has been well established from macroscopic measurements that this preparation has a large surface potassium conductance (Mathias et al., 198 S), single channel recording has consistently failed to identify a potassium channel in sufficient density to explain the macroscopic results (Jacob, 1984; Rae, 1985). The motivation for this study was to see if the potassium conductance of amphibian lens could be demonstrated using whole-cell voltage clamp recording, and to seeif the potassium conductance is similar to that in the mammalian preparations. Our major finding is that the potassium conductance is easily demonstrated and that it is different from that seen in mammalian species. In addition to this potassium current, we also noted the existence of two other currents. 2. Materials and Methods Cell Dissociation Rana pipiens were housed at room temperature and fed daily. The eyes were removed and the lenses dissectedfree from the globe using standard procedures (Cooper, Rae and Gates, 1989). The lens capsule was peeled from the posterior surface and pinned to a Sylgard disc. The fiber cell masswas then removed and the remaining epithelial sheet-capsule preparation was cleaned of any adhering fiber cell debris. The disc holding the epithelium was transferred to one well of a multi-well plate containing 0.08% trypsin (Sigma type III) and 2 mM EGTA in normal Ringer’s solution. 0 1992 Academic Press Limited
K. COOPER
862
ET AL.
TABLE I Solution composition
2.5 104 107 22.5
Normal Ringer Pipette Solution 107K 22K
MeS* Na ___-. 0 104.5
Cl
K
Solution
114 10
100
10
2.7 12.7
111.3
5 89.5
101.3
Ca
EGTA
SO,
Mg
2
0 2
0 0 1.5 1.5
0 1.5 1.5
0 1
0
1
0
1.5
* Methane sulfonate.
(A)
a a 8
L5 msec
~_____.
80 70
0 60 50a ,a ; E 3
0
40
0
30-
0 0
zoI 0
IOO0
Ooo -IO
-120
_!L--80
_ u
0
0
-40
I 40
0 Voltage
I 80
I I20
i
hV1
FIG. 1. A, Whole-cell currents in responseto a step voltage clamp protocol. The cells were bathed in normal Ringer’s solution, The voltage pulses were 40 msec in duration and went from - 140 mV to + 140 mV in 20 mV increments. The cells were held at - 60 mV between pulses. This current is the average of five repetitions of the protocol. In this and all subsequent figures, capacitance transients have been edited. B. Current-voltage relationship from (A), measured at the end of the voltage pulse. The cells remained in this solution for 30 min. At the end of this period, the disc was transferred to a well containing normal Ringer’s solution. The cells were gently triturated off the capsule using a firepolished Pasteur pipette. The solution containing the isolated cells was then transferred to a conical tube until use.
Whole-cell Recording A drop of solution containing isolated cells was placed in a custom built chamber (volume about 300 ~1) attached to the stage of an inverted Nikon Diaphot microscope. Cells typically attached within
FROG
LENS
POTASSIUM
CURRENT
5 min to a piece of microscope slide glass placed in the bottom of the chamber within 5 min. Whole-cell recording was done using standard techniques (Rae and Levis, 1984). Electrodes were made of Corning 7052 glass. After fire polishing, the electrodes had resistances of about 4.5 MQ. Recordings were made using a modified Axopatch-1B patch clamp (Axon Instruments, Foster City, CA). The data were digitized using a TL-1 A/D-D/A interface (Axon Instruments) into a modified IBM-AT computer. Data were acquired and analyzed using the pCLAMP software package (Axon Instruments). Recordings were made with a bandwidth 5 kHz and sampled every 100 psec. The whole cell time constant determined by the electrode accessresistance and the cell capacitance was typically about 160 psec. Capacitance transients were cancelled electronically. Any residual transient was removed using Drawperfect (WordPerfect Corp., Orem, UT). All experiments were performed at room temperature. The isolated cells had a mean diameter of 17.3 f0.4 pm (mean? s.E.M.; n = 47) and a mean capacitance of 11-4 + O-5 picoFarads (n = 47). Recordings were made using a modified version of the perforated patch recording technique (Rae et al., 1991). In brief, this technique uses the pore forming ant.ibiotic amphotericin-B to produce a low access resistance pathway from the recording pipette into the cell interior. The amphotericin-B is added to the pipette filling solution in a concentration of 240 ,ug ml-’ (about 2.5 x 10Y4mM). The tip of the electrode is filled to approximately 500 i&m with amphotericin-B free intracellular solution. This was necessary because amphotericin-B interferes with seal formation. Amphotericin-B takes about 10 min to diffuse to the tip and partition into the patch membrane in sufficient quantity to allow recording. The advantage of this technique over traditional whole-cell recording is that the amphotericin-B pore is only permeable to small monovalent ions. Thus, the concentrations of control molecules, like CAMP and calcium, are not altered during recording. The final accessresistance was usually about 1.5MR. Because of the low access resistance and small currents (approximately 100 PA), series resistance compensation was unnecessary (maximum voltage error under 5 mV).
863
as needed with the membrane impermeant anion methane sulfonate. The pipette solution was designed to mimic the internal composition of frog lens epithelial cells. Normal cellular impermeant anions were balanced by the membrane impermeant anion, methane sulfonate and internal calcium was buffered to a low level using EGTA. However, given that amphotericin-B is divalent impermeant. the exact value of the pipette calcium is unimportant. 3. Results Potassium Current
Figure 1(A) shows the responseof frog lens epithelial cells to a series of voltage clamp pulses. The pulses IOOr
Voltoge
(mV)
FIG. 2. Potassiumselectivity of whole-cell current. Cur-
rent-voltage relationships from a cell in three different external potassiumconcentrations.(0) 2.5 K: (V) 22.5 K:
Solutions
The composition of all solutions is given in Table I. All solutions were buffered with 5 mM Hepes. The external solutions were buffered to a pH of 7.35, and the internal solution to 7.00. All solutions had an osmolality of 22 5 mosm kg-‘. The 107K and 22K solutions are modified Ringer solutions used to test the selectivity of the whole-cell current. They were designed to have a nearly constant KC1 product to minimize cell volume changes. To maintain the constant KC1product, Cl was replaced
FIG. 3. Barium block: (0) control current-voltage relationship : (0) current-voltage relationship after addition of 5 mM barium to the bath: (0) differences between control and barium current-voltage relationships.
K. COOPER
864
ET AL.
(A)
20 msec
-__
IO00 (El 800
c
Voltage
(rnv)
4. Time-dependentcurrent. A, Current in responseto a family of voltagestepsof 100 msecduration. Pulseswent from - 120 mV to +20 mV increments.Five repetitionswere averaged.B, Current at the end of the lOO-msecpulsein (A) plotted againstpulsevoltage. FIG.
were of 40 msec duration and spanned a voltage range from - 140 to 140 mV in 20 mV increments. In general, five repetitions were averaged to produce the data. The cells were held at - 60 mV during the interpulse period. Figure l(B) shows the steady-state current-voltage relationship from Fig. l(A). The current-voltage relationship is roughly exponential, with outward currents being larger than inward currents from the same driving force. This behavior was representative of that seen in 40 of the 47 cells studied. There was no significant time dependenceto the current. The cell’s resting voltage was taken to be the zero current point on the current-voltage curve. The average resting voltage of these cells was - 56 f 4 mV (n = 47). The cell input resistance was determined from the slope of the current-voltage relationship in the vicinity of the resting voltage. The average value was 4.9 + O-6 GQ (n = 47).
To demonstrate the potassium selectivity of the resting potential, the bath solution was exchanged for a series of solutions containing varying potassium concentrations. Figure 2 shows the resulting currentvoltage relationships for external potassium concentrations of 2.5, 22.5 and 107 mM. The resting voltage shifted in the direction expected for a potassium selective membrane, and the inward current increased with increasing external potassium concentration as expected. Similar behavior was seen in five cells. The residual reversal potential in the high potassium solution represents an artifactual offset voltage. Since internal and external solutions are nearly identical, it seems unlikely that this offset was due to a residual membrane current. The possibility that this offset was due to the sodium pump was examined by adding 0.1 mM ouabain to the bath. No consistent change in the offset was seen.
FROG
LENS
POTASSIUM
CURRENT
865
I
5 msec
I
a a 0" L-
-
5 msec
FIG. 5. Current activation. A, Control current in response to 50 msec pulses from - 100 mV to + 140 mV. B, Current from same cell 15 min later. Voltage pulses go from - 140 mV to + 140 mV.
In an attempt to isolate the potassium current, the potassium channel blockers barium and tetraethylammonium (TEA) were used. TEA was found to be an ineffective blocker at concentrations up to 10 mM (data not shown). Figure 3 shows the control steady-state current-voltage relationship (open circles) and the current-voltage relationship after block by 5 mM barium (filled circles). The resting voltage shifts from - 60 mV to 0 mV in the presence of barium. The difference between these two current-voltage relationships is the potassium current of interest (triangles). The difference current reverses at a voltage very near the calcuIated potassium reversal potential (- 95 mV), and is thus a potassium current. The current is outwardly rectifying and declines at voltages above 50 mV. The currents before and after barium addition had no time dependence.Similar behavior was seenin nine other cells. Other Currents
In addition to the potassium current described
above, a time-dependent current was seen in seven of the 47 cells investigated. Figure 4(A) shows this current. It exhibits slow (about 100 msec time constant) activation kinetics. Figure 4(B) shows the steady-state current-voltage relationship from Fig. 4(A). The current shows outward rectification, becoming significant only at voltages above 0 mV. The fact that the current reversesat 0 mV could imply that this is a nonselective current. Another possibility is that the current is the sum of a non-selective leak (e.g. current through the seal) and a voltage-dependent current that does not activate until around 40 mV. The selectivity of such a current can not be determined from this experiment. In four of the ceils, the current increased dramatically during recording as shown in Fig. 5. This often coincided with a noticeable increase in cell diameter. When barium was applied, the activated current could be separated into two components. Figure 6(A) shows the current remaining after barium addition. The current-voltage relationship is presented in Fig. 6(B). The current reverses at 0 mV and has the
K. COOPER
866
ET AL.
2500 z 2002 5 150L =
0
IOO0 50-
0 C’ O0
O(W -50 -50
1 II -120
I -80
I -40
I 0 Voltage
I 40
I 80
I I 120
(mV)
FIG.6. A, Current from cell in Fig. 5 after addition of 5 mM barium. B, Current-voltagerelationship after barium. samegeneral shape as the current-voltage relationship shown in Fig. 4. Despite these similarities, the current is lacking the large tail seen in Fig. 4(A). This may represent block of the inward current by barium. It is also noteworthy that the tie course of activation is much faster than in Fig. 4(A). The difference between Fig. 5(B) and 6(A) is the current blocked by barium [Fig. 7(A)]. The steady-state current-voltage relationship is shown in Fig. 7(B). The negative reversal potential, outward rectification, and decline above 50 mV are all similar to the potassium current identified in Figs 1-3. Although the steady-state current is similar to the control potassium current, it has an early transient decay that is not seen in the control case. This may represent yet another current. 4. Discussion The properties of the cells studied here are comparable to those reported in previous work on isolated frog lens epithelial cells (Rae, 1985 ; Cooper et al., 1989). In particular, the mean resting voltage of - 56 mV agrees with that reported in several other
studies (Jacob,1984; Cooper et al.. 1986). However, it should be noted that there are several potential problems associatedwith the measurement of resting voltage using the perforated patch technique. First, the measured resting voltage (V,,,,) is only an approximation of the true resting voltage (V,). This is becausethe parallel combination of the seal resistance (R,) and the cell input resistance (R,) act as a current divider, resulting in : Vmeas= V,*R,/(R, + Rm)
It is difficult to accurately measure, I?, using the amphotericin-B technique, because the seal is in parallel with the accessresistance through the patch. The amphotericin-B partitions into the patch membrane so quickly that the access resistance is never high enough relative to R, to allow an accurate measurement of R,. A reasonable estimate of the seal resistance is about 20 GQ. Given that the input resistance of these cells is around 5 GQ, the measured resting voltage could be too low by 20% or more. In addition to this error, there are errors due to junction potentials and a Donnan potential across the ampho-
FROG
LENS
POTASSIUM
CURRENT
867
(A)
a a g- l-
5 msec
-‘“OF I
-2OO’-,
I 20
I
-80
I
-40
Voltage
I
0
I
40
I
80
I
120
I
;
(mV)
FIG. 7. A, Difference between currents in Figs S(B) and 6(A). B, Current-voltage relationship of difference current.
tericin-B (Horn and Marty, 1988). Also, whenever whole-cell recording is done, the internal concentrations of potassium, sodium, and chloride are altered, hence changing the diffusion potentials for these ions. All of these errors are hard to quantitate and thus make an absolute measurement of resting voltage difficult. The good agreement with previously published results on these cells gives us some confidence that these errors are not too extreme. The major result of this paper is the identification of the potassium current responsible for the resting voltage in frog lens epithelium. Figure 3 demonstrates the large shift in resting voltage upon addition of barium to the bathing solution. The current blocked by barium is unlike any of the potassium currents seen using whole-cell recording in mammalian lens epithelia. It is outwardly rectifying, like one of the potassium currents seen in mammalian lens, but shows none of
the characteristic time dependence (Cooper et al., 1990). The frog lens potassium current reported here is not blocked by TEA but is blocked by barium. The outwardly rectifying potassium current in human lens epithelia responds to these blockers in exactly the opposite way (Cooper et al., 1990). The rectification of this current is similar to the Goldman constant field rectification expected from the diiering concentrations of potassium on the two sides of the membrane. This can be seen in Fig. 2 where the inward current increases as external potassium is increased. The rectification appears instantaneously. This implies one of two things: either the channel open probability is voltage dependent but the gating is too fast for the recording apparatus to seethe opening process; or the channel might have a voltage independent open probability and the rectification could be an open channel property.
K. COOPER
Another interesting property of this current is the noticeable decrease in current above 50 mV. This could arise from several mechanisms. It could be due
to a voltage dependent inactivation process. If this is the case, the rate of this process is again faster than the recording apparatus can detect. It could also be due to voltage-dependent block by an internal cation such as magnesium, as is true in some types of inward
rectifiers (Nonner and Adams, 1990). Finally, it may be an artefact due to barium activation of some outwardly rectifying current. The single-channel basis of this potassium current is unknown and it is certainly possible that more than one type of potassium channel could be involved. Single channel recording from frog lens epithelia has shown the existence of a 40 pS potassium channel (Rae, Levis and Eisenberg, 1988), but its density seems too low to explain the current reported here. If this channel were open 20 % of the time, approximately 50 such channels would be necessary to explain the magnitude of the whole cell current. Given that the cells have an area of about 1000 ym2, and a patch of membrane in a single-channel experiment is on the order of 10 pm”, one would expect to seesuch a singlechannel in roughly every other patch. However, the reported density was one potassium channel in about
1000 patches. Such discrepancies between singlechannel and macroscopic current recording are not uncommon (Cooper et al., 199 1).
Reports of macroscopic measurements of potassium conductance in amphibian lens show somesimilarities to the data reported here. The exponential shape of the current and the barium sensitivity are similar to that
reported from whole frog lens (Delamere, Duncan and Paterson, 1980). Also, recent experiments in toad Iens using short circuit current measurements have identified a pH sensitive potassium conductance that may
be involved in volume regulation (Alvarez, Wolosin and Candia, 1991). This current is sensitive to barium.
In addition to the potassium current, two other currents were seen: one was time-independent and the other was a time-dependent outward rectifier. The physiological role of these currents is unclear, but if
either of them is a non-selective current in might explain why the lens resting voltage is depolarized relative to the potassium reversal potential. The barium sensitive component of the activated current has many properties similar to the potassium current, In summary,
whole-cell
recording
has allowed
identification of the major current types in isolated frog lens epithelial celIs. These include: an outwardly rectifying
time-independent
potassium current,
and
two other outwardly rectifying currents. These currents might be sufficient to explain the ionic basis of the resting voltage in the frog lens. The potassium current is distinct from any of the whole-cell recorded
potassium currents from mammalian lens epithelia.
ET AL.
Acknowledgements The authors would like to acknowledge Erika Wohlfiel for expert technical assistance in preparing this manuscript. This work was supported by NIH grants EYO3282. EY06005, EY09636 and an unrestricted award from Research to Prevent Blindness. References Alvarez, L. J., Wolosin, J. M. and Candia, 0. A. (1991). Contribution for a pH-sensitive and tonicity-sensitive K” conductance to toad translens short-circuit current. Exp. Eye Res. 52. 283-92.
Cooper, K.. Gates, P., Rae, J. L. and Dewey. J. (1990). Blectrophysiology of cultured human lens epithelial cells. 1. Membr. Biol. 117, 285-98. Cooper, K., Rae, J. L. and Dewey. J. (1991). Inwardly rectifying potassium current in mammaliam lens epithelial cells. Am. J. Physiol. 261, Cl 15x123. Cooper, K., Rae, J. L. and Gates, P. (1989). Membrane and junctional properties of dissociated frog lens epithelial cells. 1. Membr. BioI. 111, 2 15-2 7. Cooper, K., Tang, J. M., Rae, J. L. and Eisenberg,R. S. (1986). A cation channel in frog lens epithelia responsive to pressure and calcium. 1. Membr. BioZ.93, 25949. Delamere, N. A., Duncan, G. and Paterson, C. A. (1980). Characteristics of voltage-dependent conductance in the membranes of a non-excitable tissue: the amphibian lens. 1. Physiol. 308, 49-59. Duncan, G. (1969). The site of the ion restricting membranes in the toad lens. Exp. Eye Res. 8, 406-12. Guggino, W. B. (1989). Potassium and cell volume. In The Regulation of Potassium Balance (Eds Seldin, D. W. Giebisch, G.). Pp. 121-137. Raven Press, Ltd.: New York, U.S.A. Horn, R. and Marty, A. (1988). Muscarinic activation of ionic currents measured by a new whole-cell recording method. 1. Gen. Physiol. 92, 145-59. Hughes, B. A. and Steinberg, R. H. (1990). Voltagedependent currents in isolated cells of the frog retinal pigment epithelium. I. Physiol. 248, 273-97. Jacob,T. J. C. (1984). Three types of channel activity in frog lens epithelial cells. Exp. Eye Res. 38. 657-60. Mathias, R: T. (1985). Steady-statevoltages, ion fluxes, and volume regulation in syncytial tissues. Biophys. J. 48, 435-48. Mathias, R. T., Rae. J. L.. Ebihara, L. and McCarthy, R. T. (198 5). The localization of transport properties in the frog lens. Biophys. J. 48, 423-34. Rae, J. L. (1979). The electrophysiology of the crystalline lens. Curr. Topics Eye Res. 1, 3 7-90. Rae, J. L. (1985). The application of patch clamp methods of ocular epithelia. Curr. Eye Res. 4. 409-20. Rae, J. L. and Cooper. K. (1990). New techniques for the study of lens electrophysiology. Exp. Eye Res. 50, 603-6. Rae, J., Cooper, K., Gates, P. and Watsky, W. (1991). Low access resistance perforated patch recordings using amphotericin B. 1. Neurosci. Methods 37, 15-26. Rae, J. L. and Levis, R. A. (1984). Patch voltage clamp on lens epithelial cells: theory and practice. MoI. Physiol. 6, 115-62.
Shinohara, T. and Piatigorsky, J. (1977). Regulation of protein synthesis, intracellular electrolytes and cataract formation in vitro. Nature 270, 406-11.
