806
D A T A STORAGE A N D ANALYSIS
[55]
molecules provide useful models of drug-induced blocking processes. From a careful perspective, a single channel is best approached as the analog of a purified enzyme preparation in the hands of an enzymologist. The confidence gained by knowing that one is viewing a single subtype must be weighed against the possibility that the channel could have been altered in the process of patch isolation or bilayer reconstitution. As in all kinetic studies, a curve fit to a two-state scheme is contingent on the possibility that a more complex multistate system can masquerade as the simple cartoon one would like to put forward. Acknowledgments This work was supported by grants from the National Institutes of Health (AR38796 and HL38156) and an Established Investigator award from the American Heart Association.
[55] A n a l y s i s
of Sodium Channel
Tail Currents
B y GABRIEL COTA a n d CLAY M. ARMSTRONG
Introduction Analysis of tail currents has yielded much information about the functional properties of voltage-gated ion channels. Aspects of channel behavior that can be inferred from tail currents include the closing kinetics, the open-channel (or instantaneous) current-voltage relationship, and the voltage dependence of the fraction of open channels. Among other examples, the study of tail currents has been helpful in demonstrations that blocking agents can be trapped in closed channels ~-5 and that a single cell can express distinct types of Na + channels, 6 Ca 2+ channels, 7-H or K + channels, n 1C. M. Armstrong, J. Gen. Physiol. 58, 413 (1971). 2 C. M. Armstrong and S. R. Taylor, Biophys. J. 30, 473 (1980). 3 C. M. Armstrong, R. P. Swenson, and S. R. Taylor, J. Gen. Physiol. 80, 663 (1982). 4 D. Swandulla and C. M. Armstrong, Proc. Natl. Acad. Sci. U.S.A. 86, 1736 (1989). 5 R. H. Chow, J. Gen. Physiol. 98, 751 (1991). 6 W. F. Gilly and C. M. Armstrong, Nature (London) 309, 448 (1984). C. M. Armstrong and D. R. Mattes)n, Science 227, 65 (1985). s G. Cota, J. Gen. Physiol. 88, 83 (1986). 9 M. Hiriart and D. R. Mattcson, J. Gen. Physiol. 91, 617 (1988). 1oD. Swandulla and C. M. Armstrong, J. Gen. Physiol. 92, 197 (1988). 11L. Tabarcs, J. Urefia, and J. Lopez-Barneo, J. Gen. Physiol. 93, 495 (1989). 12A. Castcllano, J. Lopez-Barnco, and C. M. Armstrong, PfluegersArch. 413, 644 (1989).
METHODS IN ENZYMOLOGY, VOL. 207
Co~t © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[551
ANALYSIS OF TAIL CURRENTS
807
This chapter focuses on whole-cell patch clamp experiments in clonal pituitary GH3 cells and illustrates how the analysis of tail currents has helped us to explore some properties of Na + channels, including the kinetics of channel inactivation and the interaction of divalent and trivalent cations with the channels. GH 3 cells are derived from a rat pituitary adenoma, 13 and they have proved to be an excellent model for the study of Na + channel properties with patch clamp techniques.~4-2° The specific objectives of our analysis are (1) to determine the voltage dependence of the inactivation step; (2) to decide whether modification of channel gating by external lanthanum ion can be explained by surface charge theory; and (3) to examine the idea that calcium ion serves as a gating cofactor. In most of these experiments, gating of Na + channels has been simplified by using intracellular papain to remove inactivation. Details of the methodological procedures that we use to record Na + channel currents from GH3 cells are given in the following section. The last section summarizes the major findings. Recording of Sodium Channel Currents
Cell Culture We obtain the GH a cells from the American Type Culture Collection (Rockville, MD) and maintain them in polystyrene 25-cm2 culture flasks (Coming Glass Works, Coming, NY) using Kennetts's HY medium (Cell Center, University of Pennsylvania, Philadelphia, PA, or Hazleton Research Products, Lenexa, KS) supplemented with 5% fetal bovine serum (GIBCO, Grand Island, NY) and 1% glutamine (GIBCO). Cells are cultured in a humidified atmosphere of 5% CO2-95% air at 37 °. The maintenance culture is split every 10 days by using a brief proteolytic digestion in a dilute solution of trypsin (trypsin-EDTA; Flow Laboratories, McLean, VA) to detach the cells, replafing the monodispers~l cells at 7-fold lower density. For the electrophysiological experiments, at the time of splitting some cells are plated on slivers (11 X 2.5 ram) of glass coverslips in 35-mm ~3A. H. Tashjian, Jr., this series, Vol. 58, p. 527. 14j. M. Dubinsky and G. S. Oxford, J. Gen. Physiol. 83, 309 (1984). 15 j. M. Fernandez, A. P. Fox, and S. Krasne, J. Physiol. (London) 356, 565 (1984). t6 R. Horn and C. A. Vandenbcrg, J. Gen. Physiol. 84, 505 (1984). ~7D. R. Matteson and C. M. Armstrong, J. Gen. Physiol. 83, 371 (1984). is C. A. Vandcnberg and R. Horn, J. Gen. Physiol. 84, 535 (1984). ~9G. Cota and C. M. Armstrong, J. Gen. Physiol. 94, 213 (1989). 2o C. M. Armstrong and G. Cota, J. Gen. Physiol. 96, 1129 (1990).
808
DATA STORAGE AND ANALYSIS
[55]
plastic petri dishes. We use a subcultivation ratio of 1 : 15 and record from cells cultured for 2 - 7 days after replating. The culture medium is replenished every day.
Recording Conditions Coverslips with attached GH3 cells are transferred from culture dishes to the experimental chamber, which has a relatively small volume (-0.2 ml) and is mounted on the stage of an inverted Diaphot TMD microscope (Nikon Corporation, Tokyo, Japan). The external recording solution (see below) is continually perfused through the chamber by using a gravity-driven flow/suction arrangement. The fluid height in the chamber is controlled by adjusting the flow rate and the position of the suction tube. This tube is a 15-gauge needle with its end beveled at a 45 ° angle, covered with 1000 mesh gold screen (Ted Pella, Redding, CA). With fast flow, the solution exchange in the chamber is nearly complete in about 30 sec. The temperature of the solution in the chamber is kept at 15 ° using a controller device connected to a Peltier cooler, with a thermistor in the chamber acting as a temperature sensor. Cells are visualized at 600 × magnification and approached with firepolished pipettes containing the internal recording solution (see below). We select isolated cells that are almost spherical and 15-25/zm in diameter.
Recording Solutions The composition of the recording solutions is designed to isolate currents through Na + channels from currents carried by other cation-selective channels. We use K+-free solutions to eliminate K + currents, and we sometimes include 0.2 m M CdCI2 in the external medium in an attempt to suppress current through Ca 2+ channels. With 2 m M Ca 2+ in the external solution, the Ca 2+ current is usually small compared with the Na + current even in the absence of Cd 2+, and after papain action (see below) channel activity resistant to 1/zM external tetrodotoxin is practically absent. We use two different internal solutions in the experiments described here. The internal solution A contains (concentrations in mM) 100 NaF, 30 NaCI, 1 CaC12, and 10 EGTA-CsOH, and the internal solution B contains 30 NaCI, 9 NaF, 91 CsF, and 10 EGTA-CsOH. These solutions are supplemented with 1 mg/ml papain (see below). The composition of the external solutions is indicated in the figure legends, with concentrations in millimolar units. All solutions also contain 10 m M HEPES acid, which was neutralized to pH 7.30 with CsOH (internal solutions) or NaOH (external solutions).
[55]
ANALYSIS OF TAIL CURRENTS
809
Whole-Cell Clamping and Data Acquisition To examine the activity of Na ÷ channels, GH3 cells are subjected to whole-cell patch clamping.2~ Voltage steps are applied to the cell membrane from a holding potential of - 8 0 mV and are practically complete within 50/zsec. This relatively high time resolution for monitoring current is obtained with the combined use of a low (10 f~) feedback resistance on the head stage amplifier (an OPA-111; Burr Brown Research Corp., Tucson, AZ), low-resistance patch electrodes (see below), and "supercharging," an improved patch clamp circuit.22 Supercharging speeds the change of membrane voltage (Vm) by altering the command voltage applied to the positive input of the head stage amplifier. A 15-/zsec voltage spike of appropriate size is added to the leading edge of the command step. The spike enhances the speed with which the membrane capacitance is charged by driving current rapidly through the electrode resistance. The spike is terminated when Vmreaches the required level. We adjust the spike amplitude by watching the current transient during the first 200/~sec after a square change in command voltage. After breaking into the cell with the patch electrode, the transient has a very fast component corresponding to charging current for the stray capacitance of the electrode, as well as a slower component with a time constant equal to the product of access resistance times membrane capacitance. The appropriate amplitude of the voltage spike is determined by nulling the slower component. Spike amplitude is readjusted at frequent intervals because the access resistance normally changes during the experiment. Pulse generation and data acquisition are controlled by an LSI-11/73 computer (Scientific Micro Systems, Mountain View, CA). Membrane current signals are sampled at 10- or 20-/zsec intervals, and their linear components are subtracted out using the scaled current response to 50-mV hyperpolarizing steps.
Patch Electrodes Patch pipettes are fabricated from hard glass capillaries. We have used either aluminosilicate glass (A-M Systems, Everett, WA) or borosilicate glass (Kimax 51; Kimble Div., Owens-Illinois, Inc., Toledo, OH). Pipettes are pulled in two steps using a vertical puller (Kopf Model 700C; David Kopf Instruments, Tujunga, CA), with a coil that is two turns of l-ram 21 O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, Pfluegers Arch. 391, 85 (1981). 22 C. M. Armstrong and R. H. Chow, Biophys. J. 52, 133 (1987).
810
D A T A STORAGEA N D ANALYSIS
[55]
nichrome wire (David Kopf Instruments), shaped to 2.5 m m inside diameter and 2.3 m m length. Pipette tips are then carefully fire polished to a bullet shape using a homemade microforge. In most of our experiments, electrode resistance is between 0.4 and 0.7 M~. With these electrodes, and after papain action (see below), the access resistance is 0.5 - 1.0 MO, and the estimated series resistance error is usually smaller than 3 inV.
Removal of Inactivation by Papain To remove the fast inactivation gating of Na + channels, we apply papain (1 mg/ml; type IV, Sigma Chemical Co., St. Louis, MO) inside the cells. The enzyme is added to the internal solution, contained in the patch pipette. After break-in with the electrode, inactivation is slowly removed over the course of 10 rain, making it possible to obtain control traces of Na + currents before the enzyme acts on Na + channel gating) 9 After papain action, the cells have large and stable currents for 15-20 rain, making them an excellent preparation for many types of experiments. Inferring Sodium Channel Properties from Tail Currents It is convenient to start this section by studying Fig. l, which presents a series of Na + current traces through Na + channels with inactivation removed. The channels have activation gates that open in response to membrane depolarization and close on repolarization. The magnitude of the current depends on the number of conducting channels and on the electrochemical force that drives Na + ions through the channels. In this case the Na + equilibrium potential had a small positive value ( - 4 mV). At - 8 0 mV (the holding potential) all channels are in the gate closed state, and current is zero. In each trace, the channels are opened (activated) by a change in membrane voltage (V=) from - 8 0 mV to a depolarized level and are closed (deactivated) by the return to - 8 0 mV. In response to depolarization, inward Na + current activates with a sigmoidal time course as the number of conducting channels increases. On stepping back to - 8 0 mV the current magnitude jumps because of the sudden increase in driving force for Na + entry. The tail current then decays as the channel gates close, with a time course that can be approximated with a single exponential.
Voltage Independence of the Inactivation Step With inactivation intact, an open Na + channel can cease to conduct either because its activation gate closes (deactivation), or because its inactivation gate closes. The rate constant of channel closing is then the sum of the inactivation rate constant (k) and the deactivation rate constant (b).
[55]
ANALYSIS OF TAIL CURRENTS -60
mV
. - ;--T
.
.
.
.
.
811
.
.
--.~--
-5O -40
/ -30
..,.....,.... ......
"'"'"......•..
f • ...... ........ . ............................
/ ! l
-20 •
•.....
""'"""'"'""
"•''"'"'""-.............
................... ,.. ................... . . . _ .
/
.....
/
a s
-10 "" " " . " . " . . . , . . , . . , . . . . . . . . . . . . . . . . . , . . . , . . . . . . . . . . . . . . . . . . , . . . . . . . . . . , . . . . .
....
a*
i 2 nA
.;
J
2.5 ms FIG. I. Opening and closing of sodium channels.The traces arc wbole-cel Na+ currents
recorded from a OH3 cell after removing the inactivation gating of the Na+ channels with internal papain. Channels were opened by 10-reset steps from - 8 0 mV to the indicated voltages and were closed by the return to - 8 0 inV. The initial amplitude of the tail current recorded on repolarization is directly proportional to the number of channels with open gates at the end of the activating pulse. The time course of the tail current marks the dosing of the gates. External solution: 150 NaC1, 2 CaC12; internal solution, internal solution A (see text). We have determined k by c o m p a r i n g the channel d o t i n g kinetics before and after the proteolytic r e m o v a l of inactivation. 19 We activated the channels with a brief pulse to + 60 m V and studied the time course of channel closing on changing V= to a second, usually m o r e negative level. W e found that channels no longer close at or positive to - 20 m V when the inactivation gate has been removed, which indicates that d o s i n g o f intact channels at these voltages results exclusively f r o m inactivation. T h e time course o f inactivation at - 2 0 m V can be c o m p a r e d with that at 0, + 40,
812
DATA STORAGE AND ANALYSIS
~. pc.~e.le~.~.. • • " " . . . . .
• ......
• ...................
-20 mV, a j "
/
[55]
•
.
1 nP
/
;
2 ms
op %
,..,
40
~"'~'w~,~ .....................................
\ 60 ,~, "IP~'u~,w.%~ . . . . .
..,.,..
.....
.,.,
....................
FIG. 2. The inactivation step is not voltage dependent. The traces are Na + currents recorded after 0.4-msec pulses to +60 mV, before removal of inactivation with internal papain. To analyze the time course of channel closing at different voltages, Vm was first stepped from - 8 0 to + 60 mV, then maintained at this value (lower mace) or changed to +40, 0, or - 2 0 inV. All of the decay in current magnitude at these voltages results from inactivation. This is clear because current magnitude shows no decay at or positive to - 2 0 rnV after removing inactivation. There is no signitieant effect of Vm on the rate of channel inactivation. External solution: 75 NaCI, 75 choline chloride, 2 CaCI 2, 0.2 CdC12; internal solution, internal solution B (see text).
and + 60 mV in Fig. 2. At every voltage, channel inactivation was well fit by a single exponential. The rate constant (k) was 1.08 msec -~ at - 2 0 mV, 1.07 msec -~ at 0 mV, 1.03 msec -! at + 4 0 mV, and 1.06 msec -~ at + 6 0 mV. The effect of Vm on k is thus negligible. These results show unequivocally that the rate constant for the transition of an open Na + channel into the inactivated state has no significant voltage dependence over a wide range of membrane voltages.
Modification of Channel Gating by Lanthanum Divalent and trivalent cations have strong effects on Na + channel function. They shift the gating behavior of the channel along the voltage axis, 2a and Ca 2+ (and presumably other ions as well) causes voltage-dependent block of the channels.24-29 It is usually considered that alteration of
[55]
ANALYSIS OF TAIL CURRENTS
813
gating and blocking are two separate actions, with the effects on gating explained in terms of surface charge theory. In its uniform surface charge version (the version commonly considered), the surface charge theory predicts that all aspects of channel gating should be affected equally. We have tested this prediction in inactivationless Na + channels by studying the changes in gating induced by substitution of La 3+ for Ca 2+ in the external medium. 2° To quantify La 3+ effects, we recorded the Na + currents caused by 10-msec activating pulses to various membrane potentials followed by repolarization to - 80 mV, as in Fig. 1. We then empirically fitted the late phase of opening of the channels at every voltage with a single exponential, determined the opening (activation) rate (a), and plotted this rate as a function of V,,. We also determined fraction o p e n - V= curves by plotting the initial amplitude of the tail current at - 80 mV as a function of Vm during the activating pulse that preceded the tail measurement. Tail current amplitude is directly proportional to the number of channels that have open gates at the end of the activating pulse. In addition, we determined the voltage dependence of the dosing (deactivation) rate (b), from experiments similar to that in Fig. 2. The three measurable parameters of gating, namely, activation rate, deactivation rate, and the midpoint of the fraction open-voltage relation, were determined in the presence of different external La 3+ concentrations (from 5 g M to 4 mM), and the shift of each along the voltage axis, relative to their values in 2 m M Ca 2+, was quantified. Table I presents results from a representative experiment. It is clear that the three parameters are not equally shifted, as the uniform surface charge theory predicts. This discrepancy may be resolvable by invoking a nonuniform charge distribution. However, at low (5 or 10 #M) La ~+ concentrations, not only are the shifts of opening and dosing kinetics different in size, but they are of opposite sign. This seems impossible to explain by any modification of the surface charge theory. Like Ca 2+ (see below), La 3+ also blocks Na ÷ channels at negative voltages, an action that lies outside of the surface charge theory. 23 B. HiUe, "Ionic Channels of Excitable Membranes." Sinauer, Sunderland, Massachusetts, 1984. 24 A. M. Woodhull, J. Gen. Physiol. 61,687 (1973). 2s R. E. Taylor, C. M. Armstrong~ and F. Bezanilla, Biophys. J. 16, 27a (1976). 26 D. Yamamoto, J. Z. Yeh, and T. Narahashi, Biophys. J. 45, 337 (1984). 27 G. N. Mozhayeva, A. P. Naumov, and E. D. Nosyreva, Gen. Physiol. Biophys. 4, 425 (1985). 2s S. Cukierman, W. C. Zinkand, R. J. French, and B. K. Krueger, J. Gen. Physiol. 92, 431 (1988). 29 B. Nilius, J. Physiol. (London) 399, 537 (1988).
814
DATA STORAGE AND ANALYSIS
[55]
TABLE I LANTHANUM-INDUCEDSHIFTS OF THREE PARAMETERS OF SODIUM CHANNEL GATINGa
[La3+]
A
Open
B
5/tM 10//M 4 mM
11.5 16.5 53.0
-1.0 3.8 44.5
-7.5 -4.8 30.0
Opening rate (A), closing rate (B), and the midpoint of the fraction open-Vm curve (Open) (see text). Voltage shifts are in miUivolts. Recording solutions were as in Fig. 1, except that external Ca 2+ was replaced by the indicated La 3+ concentration.
Calcium May Be a Gating Cofactor Results presented above suggest that a new theory of di- and trivalent cation action is needed, and we are attempting to develop one that relates the blocking and gating effects of Ca 2+ and other multivalent ions on Na + channels. Specifically, we propose that both actions, blocking and "shifting," are related to Ca 2+ entry into the Na + channels. Our proposal is based on the finding of a close correlation between Ca 2+ block and the effects of Ca :+ on gating, 3° as described below. Calcium block was analyzed by determining instantaneous I - V (IIV) curves. The IIV curve is obtained by activating the channels with a large depolarization and then, when most of them are open, changing V,, and measuring the current (i.e., the tail amplitude) at the new voltage before, ideally, the gates of any of the channels have closed. Current is then plotted as a function of V,, in the second step. The method depends on the fact that opening and closing of the activation gates is relatively slow. The blocking reaction, on the other hand, is effectively instantaneous and cannot be time resolved. The IIV curve in the presence of 2 m M C a 2+ was nearly linear between - 3 0 and + 30 mV, but below - 3 0 mV the curve became sublinear. The curvature of negative voltage is due to the blocking action of Ca :+ and was accentuated at higher Ca 2+ concentration. From the IIV curves it is possible to estimate the fraction of the channels that are blocked at any Ca 2+ concentration and I'm, as shown in Fig. 3A. Alterations of Na + channel gating by Ca 2+ were quantified by deter3o C. M. Armstrong and G. CoLa, Proc Natl. Acad. Sci. U.S.A, 88, 6528 (1991).
[55]
ANALYSIS OF TAIL CURRENTS
A
815
Froctlon blocked
1.0 0.8
k
0.6
l o c= e . ~ . e . . " e~
0.4 2 Co 0 . . . . . ~ 0 ~ 0 ~ 0 ~ ,
,
,
-90
-70
-50
0.2 o.....Q
0.0
!
-10
-30
Vm (my) Shift
(mV)
4O 50 Co @ 30 20 Co / 20 10 0
0.0
!
0.2
0.4 0.6 Froction blocked
0.8
1.0
FIG. 3. (A) Voltage dependence of the fraction of sodium channels that are blocked by Ca 2+, as derived from IIV curves (see text). (B) Correlation between the effect of Ca'+ on Na + channel gating and Ca2+ block. The Ca2+-induced shitt of the midpoint of the fraction open- V~ curve, relative to the value in 2 m M Ca 2+, is plotted as a function of the fraction of Na + channels that are Ca2+ blocked at - 8 0 mV. External solutions with the Ca2+ concentration (raM) indicated were appropriate mixtures of the following two solutions: "50 Ca" solution; 50 CaC12, 80 NaC1; "0 Ca" solution; 80 NaC1 and sucrose to raise the osmolarity to 300 mosmol. The internal solution was internal solution A. Raised Ca2+ increases the blocked fraction of channels. This action seems to be closely related to a stabilization of the closed conformation of the channel.
mining the midpoint of the fraction o p e n - Vm curve and the deactivation rate constant. The external Ca 2+ concentration ranged from 2 to 50 mM. At every [Ca2+], normalized activation curves relating the fraction of channels with open gates to voltage were obtained by dividing the Na +
816
DATASTORAGEANDANALYSIS
[56]
conductance at the end of 10-msec activating pulses by the corresponding fraction of the channels that were not Ca2+ blocked. Figure 3B shows that the shift of the midpoint of the fraction open- Vm curve is almost directly proportional to the fraction blocked at - 8 0 mV. Curves using block at - 5 0 , - 6 0 , and - 7 0 mV gave similar correlation lines. There was also a close correlation between the degree of block at a given voltage and the rate at which the channel gates close. Because blocking is obviously related to the rate at which Ca2+ ions are entering the channels, these correlations suggest strongly that "shifting" and closing are also so related. Calcium ion entry into Na+ channels thus seems to play a part in the opening and dosing of these channels. Although details of this role of Ca2+ are likely to be complicated, we tentatively suggest that the channel activation gates close stably when the channel is occupied by Ca2+ (or a suitable ion substitute), as is the case for potassium channels in squid neurons, 3~,32 so that normally the state of the Na+ channel on opening of the gates is Ca2+ blocked, and block is a step in the closing pathway. 3~ C. M. Armstrong and D. R. Matteson, J. Gen. Physiol. 87, 817 (1986). 32 C. M. Armstrong and J. Lopez-Barneo, Science 236, 712 (1987).
[56] C a l c u l a t i o n o f I o n C u r r e n t s f r o m E n e r g y P r o f i l e s a n d E n e r g y Profiles f r o m I o n C u r r e n t s in M u l t i b a r r i e r , Multisite, Multioccupancy Channel Model
By OSVALDO
ALVAREZ, A L F R E D O VILLARROEL,
and G E O R G E EISENMAN
Introduction In the quest for finding the molecular basis of ion transport through membrane channels, the connection between structure and function is the free energy profile of the ion in the pathway.',2 This energy profile can be calculated if the structure is known, using the tools of theoretical chemistry2-6 On the other hand, the systematic measurements of the B. Hille, 3". Gen. Physiol. 66, 535 (1975). 2 G. Eisenman and J. A. Dalai, Annu. Rev. Biophys. Biophys. Chem. 16, 205 (1987). 3 S. Sung and P. Jordan, Biophys. ,!. 51, 661 (1987). 4 G. Eisenman, A. Oberhauser, and F. Bezanilla, in "Transport through Membranes: Carriers, Channels and Pumps" (A. Pullman, J. Jortner, and B. Pullman, eds.), p. 27. Kluwer, Academic Publishers, Dordrecht, The Netherlands, 1988. 5 S. Furois-Courbin and A. Pullman, in "Transport through Membranes: Carriers, Channels
METHODSIN ENZYMOLOGY,VOL. 207
Copyright© 1992by AcademicPress,Inc. Allrightsof~'l~'oduclionin any formreserved.
D A T A STORAGE A N D ANALYSIS
[55]
molecules provide useful models of drug-induced blocking processes. From a careful perspective, a single channel is best approached as the analog of a purified enzyme preparation in the hands of an enzymologist. The confidence gained by knowing that one is viewing a single subtype must be weighed against the possibility that the channel could have been altered in the process of patch isolation or bilayer reconstitution. As in all kinetic studies, a curve fit to a two-state scheme is contingent on the possibility that a more complex multistate system can masquerade as the simple cartoon one would like to put forward. Acknowledgments This work was supported by grants from the National Institutes of Health (AR38796 and HL38156) and an Established Investigator award from the American Heart Association.
[55] A n a l y s i s
of Sodium Channel
Tail Currents
B y GABRIEL COTA a n d CLAY M. ARMSTRONG
Introduction Analysis of tail currents has yielded much information about the functional properties of voltage-gated ion channels. Aspects of channel behavior that can be inferred from tail currents include the closing kinetics, the open-channel (or instantaneous) current-voltage relationship, and the voltage dependence of the fraction of open channels. Among other examples, the study of tail currents has been helpful in demonstrations that blocking agents can be trapped in closed channels ~-5 and that a single cell can express distinct types of Na + channels, 6 Ca 2+ channels, 7-H or K + channels, n 1C. M. Armstrong, J. Gen. Physiol. 58, 413 (1971). 2 C. M. Armstrong and S. R. Taylor, Biophys. J. 30, 473 (1980). 3 C. M. Armstrong, R. P. Swenson, and S. R. Taylor, J. Gen. Physiol. 80, 663 (1982). 4 D. Swandulla and C. M. Armstrong, Proc. Natl. Acad. Sci. U.S.A. 86, 1736 (1989). 5 R. H. Chow, J. Gen. Physiol. 98, 751 (1991). 6 W. F. Gilly and C. M. Armstrong, Nature (London) 309, 448 (1984). C. M. Armstrong and D. R. Mattes)n, Science 227, 65 (1985). s G. Cota, J. Gen. Physiol. 88, 83 (1986). 9 M. Hiriart and D. R. Mattcson, J. Gen. Physiol. 91, 617 (1988). 1oD. Swandulla and C. M. Armstrong, J. Gen. Physiol. 92, 197 (1988). 11L. Tabarcs, J. Urefia, and J. Lopez-Barneo, J. Gen. Physiol. 93, 495 (1989). 12A. Castcllano, J. Lopez-Barnco, and C. M. Armstrong, PfluegersArch. 413, 644 (1989).
METHODS IN ENZYMOLOGY, VOL. 207
Co~t © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[551
ANALYSIS OF TAIL CURRENTS
807
This chapter focuses on whole-cell patch clamp experiments in clonal pituitary GH3 cells and illustrates how the analysis of tail currents has helped us to explore some properties of Na + channels, including the kinetics of channel inactivation and the interaction of divalent and trivalent cations with the channels. GH 3 cells are derived from a rat pituitary adenoma, 13 and they have proved to be an excellent model for the study of Na + channel properties with patch clamp techniques.~4-2° The specific objectives of our analysis are (1) to determine the voltage dependence of the inactivation step; (2) to decide whether modification of channel gating by external lanthanum ion can be explained by surface charge theory; and (3) to examine the idea that calcium ion serves as a gating cofactor. In most of these experiments, gating of Na + channels has been simplified by using intracellular papain to remove inactivation. Details of the methodological procedures that we use to record Na + channel currents from GH3 cells are given in the following section. The last section summarizes the major findings. Recording of Sodium Channel Currents
Cell Culture We obtain the GH a cells from the American Type Culture Collection (Rockville, MD) and maintain them in polystyrene 25-cm2 culture flasks (Coming Glass Works, Coming, NY) using Kennetts's HY medium (Cell Center, University of Pennsylvania, Philadelphia, PA, or Hazleton Research Products, Lenexa, KS) supplemented with 5% fetal bovine serum (GIBCO, Grand Island, NY) and 1% glutamine (GIBCO). Cells are cultured in a humidified atmosphere of 5% CO2-95% air at 37 °. The maintenance culture is split every 10 days by using a brief proteolytic digestion in a dilute solution of trypsin (trypsin-EDTA; Flow Laboratories, McLean, VA) to detach the cells, replafing the monodispers~l cells at 7-fold lower density. For the electrophysiological experiments, at the time of splitting some cells are plated on slivers (11 X 2.5 ram) of glass coverslips in 35-mm ~3A. H. Tashjian, Jr., this series, Vol. 58, p. 527. 14j. M. Dubinsky and G. S. Oxford, J. Gen. Physiol. 83, 309 (1984). 15 j. M. Fernandez, A. P. Fox, and S. Krasne, J. Physiol. (London) 356, 565 (1984). t6 R. Horn and C. A. Vandenbcrg, J. Gen. Physiol. 84, 505 (1984). ~7D. R. Matteson and C. M. Armstrong, J. Gen. Physiol. 83, 371 (1984). is C. A. Vandcnberg and R. Horn, J. Gen. Physiol. 84, 535 (1984). ~9G. Cota and C. M. Armstrong, J. Gen. Physiol. 94, 213 (1989). 2o C. M. Armstrong and G. Cota, J. Gen. Physiol. 96, 1129 (1990).
808
DATA STORAGE AND ANALYSIS
[55]
plastic petri dishes. We use a subcultivation ratio of 1 : 15 and record from cells cultured for 2 - 7 days after replating. The culture medium is replenished every day.
Recording Conditions Coverslips with attached GH3 cells are transferred from culture dishes to the experimental chamber, which has a relatively small volume (-0.2 ml) and is mounted on the stage of an inverted Diaphot TMD microscope (Nikon Corporation, Tokyo, Japan). The external recording solution (see below) is continually perfused through the chamber by using a gravity-driven flow/suction arrangement. The fluid height in the chamber is controlled by adjusting the flow rate and the position of the suction tube. This tube is a 15-gauge needle with its end beveled at a 45 ° angle, covered with 1000 mesh gold screen (Ted Pella, Redding, CA). With fast flow, the solution exchange in the chamber is nearly complete in about 30 sec. The temperature of the solution in the chamber is kept at 15 ° using a controller device connected to a Peltier cooler, with a thermistor in the chamber acting as a temperature sensor. Cells are visualized at 600 × magnification and approached with firepolished pipettes containing the internal recording solution (see below). We select isolated cells that are almost spherical and 15-25/zm in diameter.
Recording Solutions The composition of the recording solutions is designed to isolate currents through Na + channels from currents carried by other cation-selective channels. We use K+-free solutions to eliminate K + currents, and we sometimes include 0.2 m M CdCI2 in the external medium in an attempt to suppress current through Ca 2+ channels. With 2 m M Ca 2+ in the external solution, the Ca 2+ current is usually small compared with the Na + current even in the absence of Cd 2+, and after papain action (see below) channel activity resistant to 1/zM external tetrodotoxin is practically absent. We use two different internal solutions in the experiments described here. The internal solution A contains (concentrations in mM) 100 NaF, 30 NaCI, 1 CaC12, and 10 EGTA-CsOH, and the internal solution B contains 30 NaCI, 9 NaF, 91 CsF, and 10 EGTA-CsOH. These solutions are supplemented with 1 mg/ml papain (see below). The composition of the external solutions is indicated in the figure legends, with concentrations in millimolar units. All solutions also contain 10 m M HEPES acid, which was neutralized to pH 7.30 with CsOH (internal solutions) or NaOH (external solutions).
[55]
ANALYSIS OF TAIL CURRENTS
809
Whole-Cell Clamping and Data Acquisition To examine the activity of Na ÷ channels, GH3 cells are subjected to whole-cell patch clamping.2~ Voltage steps are applied to the cell membrane from a holding potential of - 8 0 mV and are practically complete within 50/zsec. This relatively high time resolution for monitoring current is obtained with the combined use of a low (10 f~) feedback resistance on the head stage amplifier (an OPA-111; Burr Brown Research Corp., Tucson, AZ), low-resistance patch electrodes (see below), and "supercharging," an improved patch clamp circuit.22 Supercharging speeds the change of membrane voltage (Vm) by altering the command voltage applied to the positive input of the head stage amplifier. A 15-/zsec voltage spike of appropriate size is added to the leading edge of the command step. The spike enhances the speed with which the membrane capacitance is charged by driving current rapidly through the electrode resistance. The spike is terminated when Vmreaches the required level. We adjust the spike amplitude by watching the current transient during the first 200/~sec after a square change in command voltage. After breaking into the cell with the patch electrode, the transient has a very fast component corresponding to charging current for the stray capacitance of the electrode, as well as a slower component with a time constant equal to the product of access resistance times membrane capacitance. The appropriate amplitude of the voltage spike is determined by nulling the slower component. Spike amplitude is readjusted at frequent intervals because the access resistance normally changes during the experiment. Pulse generation and data acquisition are controlled by an LSI-11/73 computer (Scientific Micro Systems, Mountain View, CA). Membrane current signals are sampled at 10- or 20-/zsec intervals, and their linear components are subtracted out using the scaled current response to 50-mV hyperpolarizing steps.
Patch Electrodes Patch pipettes are fabricated from hard glass capillaries. We have used either aluminosilicate glass (A-M Systems, Everett, WA) or borosilicate glass (Kimax 51; Kimble Div., Owens-Illinois, Inc., Toledo, OH). Pipettes are pulled in two steps using a vertical puller (Kopf Model 700C; David Kopf Instruments, Tujunga, CA), with a coil that is two turns of l-ram 21 O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, Pfluegers Arch. 391, 85 (1981). 22 C. M. Armstrong and R. H. Chow, Biophys. J. 52, 133 (1987).
810
D A T A STORAGEA N D ANALYSIS
[55]
nichrome wire (David Kopf Instruments), shaped to 2.5 m m inside diameter and 2.3 m m length. Pipette tips are then carefully fire polished to a bullet shape using a homemade microforge. In most of our experiments, electrode resistance is between 0.4 and 0.7 M~. With these electrodes, and after papain action (see below), the access resistance is 0.5 - 1.0 MO, and the estimated series resistance error is usually smaller than 3 inV.
Removal of Inactivation by Papain To remove the fast inactivation gating of Na + channels, we apply papain (1 mg/ml; type IV, Sigma Chemical Co., St. Louis, MO) inside the cells. The enzyme is added to the internal solution, contained in the patch pipette. After break-in with the electrode, inactivation is slowly removed over the course of 10 rain, making it possible to obtain control traces of Na + currents before the enzyme acts on Na + channel gating) 9 After papain action, the cells have large and stable currents for 15-20 rain, making them an excellent preparation for many types of experiments. Inferring Sodium Channel Properties from Tail Currents It is convenient to start this section by studying Fig. l, which presents a series of Na + current traces through Na + channels with inactivation removed. The channels have activation gates that open in response to membrane depolarization and close on repolarization. The magnitude of the current depends on the number of conducting channels and on the electrochemical force that drives Na + ions through the channels. In this case the Na + equilibrium potential had a small positive value ( - 4 mV). At - 8 0 mV (the holding potential) all channels are in the gate closed state, and current is zero. In each trace, the channels are opened (activated) by a change in membrane voltage (V=) from - 8 0 mV to a depolarized level and are closed (deactivated) by the return to - 8 0 mV. In response to depolarization, inward Na + current activates with a sigmoidal time course as the number of conducting channels increases. On stepping back to - 8 0 mV the current magnitude jumps because of the sudden increase in driving force for Na + entry. The tail current then decays as the channel gates close, with a time course that can be approximated with a single exponential.
Voltage Independence of the Inactivation Step With inactivation intact, an open Na + channel can cease to conduct either because its activation gate closes (deactivation), or because its inactivation gate closes. The rate constant of channel closing is then the sum of the inactivation rate constant (k) and the deactivation rate constant (b).
[55]
ANALYSIS OF TAIL CURRENTS -60
mV
. - ;--T
.
.
.
.
.
811
.
.
--.~--
-5O -40
/ -30
..,.....,.... ......
"'"'"......•..
f • ...... ........ . ............................
/ ! l
-20 •
•.....
""'"""'"'""
"•''"'"'""-.............
................... ,.. ................... . . . _ .
/
.....
/
a s
-10 "" " " . " . " . . . , . . , . . , . . . . . . . . . . . . . . . . . , . . . , . . . . . . . . . . . . . . . . . . , . . . . . . . . . . , . . . . .
....
a*
i 2 nA
.;
J
2.5 ms FIG. I. Opening and closing of sodium channels.The traces arc wbole-cel Na+ currents
recorded from a OH3 cell after removing the inactivation gating of the Na+ channels with internal papain. Channels were opened by 10-reset steps from - 8 0 mV to the indicated voltages and were closed by the return to - 8 0 inV. The initial amplitude of the tail current recorded on repolarization is directly proportional to the number of channels with open gates at the end of the activating pulse. The time course of the tail current marks the dosing of the gates. External solution: 150 NaC1, 2 CaC12; internal solution, internal solution A (see text). We have determined k by c o m p a r i n g the channel d o t i n g kinetics before and after the proteolytic r e m o v a l of inactivation. 19 We activated the channels with a brief pulse to + 60 m V and studied the time course of channel closing on changing V= to a second, usually m o r e negative level. W e found that channels no longer close at or positive to - 20 m V when the inactivation gate has been removed, which indicates that d o s i n g o f intact channels at these voltages results exclusively f r o m inactivation. T h e time course o f inactivation at - 2 0 m V can be c o m p a r e d with that at 0, + 40,
812
DATA STORAGE AND ANALYSIS
~. pc.~e.le~.~.. • • " " . . . . .
• ......
• ...................
-20 mV, a j "
/
[55]
•
.
1 nP
/
;
2 ms
op %
,..,
40
~"'~'w~,~ .....................................
\ 60 ,~, "IP~'u~,w.%~ . . . . .
..,.,..
.....
.,.,
....................
FIG. 2. The inactivation step is not voltage dependent. The traces are Na + currents recorded after 0.4-msec pulses to +60 mV, before removal of inactivation with internal papain. To analyze the time course of channel closing at different voltages, Vm was first stepped from - 8 0 to + 60 mV, then maintained at this value (lower mace) or changed to +40, 0, or - 2 0 inV. All of the decay in current magnitude at these voltages results from inactivation. This is clear because current magnitude shows no decay at or positive to - 2 0 rnV after removing inactivation. There is no signitieant effect of Vm on the rate of channel inactivation. External solution: 75 NaCI, 75 choline chloride, 2 CaCI 2, 0.2 CdC12; internal solution, internal solution B (see text).
and + 60 mV in Fig. 2. At every voltage, channel inactivation was well fit by a single exponential. The rate constant (k) was 1.08 msec -~ at - 2 0 mV, 1.07 msec -~ at 0 mV, 1.03 msec -! at + 4 0 mV, and 1.06 msec -~ at + 6 0 mV. The effect of Vm on k is thus negligible. These results show unequivocally that the rate constant for the transition of an open Na + channel into the inactivated state has no significant voltage dependence over a wide range of membrane voltages.
Modification of Channel Gating by Lanthanum Divalent and trivalent cations have strong effects on Na + channel function. They shift the gating behavior of the channel along the voltage axis, 2a and Ca 2+ (and presumably other ions as well) causes voltage-dependent block of the channels.24-29 It is usually considered that alteration of
[55]
ANALYSIS OF TAIL CURRENTS
813
gating and blocking are two separate actions, with the effects on gating explained in terms of surface charge theory. In its uniform surface charge version (the version commonly considered), the surface charge theory predicts that all aspects of channel gating should be affected equally. We have tested this prediction in inactivationless Na + channels by studying the changes in gating induced by substitution of La 3+ for Ca 2+ in the external medium. 2° To quantify La 3+ effects, we recorded the Na + currents caused by 10-msec activating pulses to various membrane potentials followed by repolarization to - 80 mV, as in Fig. 1. We then empirically fitted the late phase of opening of the channels at every voltage with a single exponential, determined the opening (activation) rate (a), and plotted this rate as a function of V,,. We also determined fraction o p e n - V= curves by plotting the initial amplitude of the tail current at - 80 mV as a function of Vm during the activating pulse that preceded the tail measurement. Tail current amplitude is directly proportional to the number of channels that have open gates at the end of the activating pulse. In addition, we determined the voltage dependence of the dosing (deactivation) rate (b), from experiments similar to that in Fig. 2. The three measurable parameters of gating, namely, activation rate, deactivation rate, and the midpoint of the fraction open-voltage relation, were determined in the presence of different external La 3+ concentrations (from 5 g M to 4 mM), and the shift of each along the voltage axis, relative to their values in 2 m M Ca 2+, was quantified. Table I presents results from a representative experiment. It is clear that the three parameters are not equally shifted, as the uniform surface charge theory predicts. This discrepancy may be resolvable by invoking a nonuniform charge distribution. However, at low (5 or 10 #M) La ~+ concentrations, not only are the shifts of opening and dosing kinetics different in size, but they are of opposite sign. This seems impossible to explain by any modification of the surface charge theory. Like Ca 2+ (see below), La 3+ also blocks Na ÷ channels at negative voltages, an action that lies outside of the surface charge theory. 23 B. HiUe, "Ionic Channels of Excitable Membranes." Sinauer, Sunderland, Massachusetts, 1984. 24 A. M. Woodhull, J. Gen. Physiol. 61,687 (1973). 2s R. E. Taylor, C. M. Armstrong~ and F. Bezanilla, Biophys. J. 16, 27a (1976). 26 D. Yamamoto, J. Z. Yeh, and T. Narahashi, Biophys. J. 45, 337 (1984). 27 G. N. Mozhayeva, A. P. Naumov, and E. D. Nosyreva, Gen. Physiol. Biophys. 4, 425 (1985). 2s S. Cukierman, W. C. Zinkand, R. J. French, and B. K. Krueger, J. Gen. Physiol. 92, 431 (1988). 29 B. Nilius, J. Physiol. (London) 399, 537 (1988).
814
DATA STORAGE AND ANALYSIS
[55]
TABLE I LANTHANUM-INDUCEDSHIFTS OF THREE PARAMETERS OF SODIUM CHANNEL GATINGa
[La3+]
A
Open
B
5/tM 10//M 4 mM
11.5 16.5 53.0
-1.0 3.8 44.5
-7.5 -4.8 30.0
Opening rate (A), closing rate (B), and the midpoint of the fraction open-Vm curve (Open) (see text). Voltage shifts are in miUivolts. Recording solutions were as in Fig. 1, except that external Ca 2+ was replaced by the indicated La 3+ concentration.
Calcium May Be a Gating Cofactor Results presented above suggest that a new theory of di- and trivalent cation action is needed, and we are attempting to develop one that relates the blocking and gating effects of Ca 2+ and other multivalent ions on Na + channels. Specifically, we propose that both actions, blocking and "shifting," are related to Ca 2+ entry into the Na + channels. Our proposal is based on the finding of a close correlation between Ca 2+ block and the effects of Ca :+ on gating, 3° as described below. Calcium block was analyzed by determining instantaneous I - V (IIV) curves. The IIV curve is obtained by activating the channels with a large depolarization and then, when most of them are open, changing V,, and measuring the current (i.e., the tail amplitude) at the new voltage before, ideally, the gates of any of the channels have closed. Current is then plotted as a function of V,, in the second step. The method depends on the fact that opening and closing of the activation gates is relatively slow. The blocking reaction, on the other hand, is effectively instantaneous and cannot be time resolved. The IIV curve in the presence of 2 m M C a 2+ was nearly linear between - 3 0 and + 30 mV, but below - 3 0 mV the curve became sublinear. The curvature of negative voltage is due to the blocking action of Ca :+ and was accentuated at higher Ca 2+ concentration. From the IIV curves it is possible to estimate the fraction of the channels that are blocked at any Ca 2+ concentration and I'm, as shown in Fig. 3A. Alterations of Na + channel gating by Ca 2+ were quantified by deter3o C. M. Armstrong and G. CoLa, Proc Natl. Acad. Sci. U.S.A, 88, 6528 (1991).
[55]
ANALYSIS OF TAIL CURRENTS
A
815
Froctlon blocked
1.0 0.8
k
0.6
l o c= e . ~ . e . . " e~
0.4 2 Co 0 . . . . . ~ 0 ~ 0 ~ 0 ~ ,
,
,
-90
-70
-50
0.2 o.....Q
0.0
!
-10
-30
Vm (my) Shift
(mV)
4O 50 Co @ 30 20 Co / 20 10 0
0.0
!
0.2
0.4 0.6 Froction blocked
0.8
1.0
FIG. 3. (A) Voltage dependence of the fraction of sodium channels that are blocked by Ca 2+, as derived from IIV curves (see text). (B) Correlation between the effect of Ca'+ on Na + channel gating and Ca2+ block. The Ca2+-induced shitt of the midpoint of the fraction open- V~ curve, relative to the value in 2 m M Ca 2+, is plotted as a function of the fraction of Na + channels that are Ca2+ blocked at - 8 0 mV. External solutions with the Ca2+ concentration (raM) indicated were appropriate mixtures of the following two solutions: "50 Ca" solution; 50 CaC12, 80 NaC1; "0 Ca" solution; 80 NaC1 and sucrose to raise the osmolarity to 300 mosmol. The internal solution was internal solution A. Raised Ca2+ increases the blocked fraction of channels. This action seems to be closely related to a stabilization of the closed conformation of the channel.
mining the midpoint of the fraction o p e n - Vm curve and the deactivation rate constant. The external Ca 2+ concentration ranged from 2 to 50 mM. At every [Ca2+], normalized activation curves relating the fraction of channels with open gates to voltage were obtained by dividing the Na +
816
DATASTORAGEANDANALYSIS
[56]
conductance at the end of 10-msec activating pulses by the corresponding fraction of the channels that were not Ca2+ blocked. Figure 3B shows that the shift of the midpoint of the fraction open- Vm curve is almost directly proportional to the fraction blocked at - 8 0 mV. Curves using block at - 5 0 , - 6 0 , and - 7 0 mV gave similar correlation lines. There was also a close correlation between the degree of block at a given voltage and the rate at which the channel gates close. Because blocking is obviously related to the rate at which Ca2+ ions are entering the channels, these correlations suggest strongly that "shifting" and closing are also so related. Calcium ion entry into Na+ channels thus seems to play a part in the opening and dosing of these channels. Although details of this role of Ca2+ are likely to be complicated, we tentatively suggest that the channel activation gates close stably when the channel is occupied by Ca2+ (or a suitable ion substitute), as is the case for potassium channels in squid neurons, 3~,32 so that normally the state of the Na+ channel on opening of the gates is Ca2+ blocked, and block is a step in the closing pathway. 3~ C. M. Armstrong and D. R. Matteson, J. Gen. Physiol. 87, 817 (1986). 32 C. M. Armstrong and J. Lopez-Barneo, Science 236, 712 (1987).
[56] C a l c u l a t i o n o f I o n C u r r e n t s f r o m E n e r g y P r o f i l e s a n d E n e r g y Profiles f r o m I o n C u r r e n t s in M u l t i b a r r i e r , Multisite, Multioccupancy Channel Model
By OSVALDO
ALVAREZ, A L F R E D O VILLARROEL,
and G E O R G E EISENMAN
Introduction In the quest for finding the molecular basis of ion transport through membrane channels, the connection between structure and function is the free energy profile of the ion in the pathway.',2 This energy profile can be calculated if the structure is known, using the tools of theoretical chemistry2-6 On the other hand, the systematic measurements of the B. Hille, 3". Gen. Physiol. 66, 535 (1975). 2 G. Eisenman and J. A. Dalai, Annu. Rev. Biophys. Biophys. Chem. 16, 205 (1987). 3 S. Sung and P. Jordan, Biophys. ,!. 51, 661 (1987). 4 G. Eisenman, A. Oberhauser, and F. Bezanilla, in "Transport through Membranes: Carriers, Channels and Pumps" (A. Pullman, J. Jortner, and B. Pullman, eds.), p. 27. Kluwer, Academic Publishers, Dordrecht, The Netherlands, 1988. 5 S. Furois-Courbin and A. Pullman, in "Transport through Membranes: Carriers, Channels
METHODSIN ENZYMOLOGY,VOL. 207
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