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Net charge transport during sodium-dependent calcium extrusion in isolated salamander rod outer segments.

The light-sensitive current and the current associated with the extrusion of internal Ca2+ in exchange for external Na+ have been recorded from detached rod outer segments from the salamander retina by the use of the whole-cell voltage clamp technique. No significant current-carrying mechanisms are present in the outer segment membrane apart from the light-sensitive conductance and the Na:Ca,K exchange, and exchange currents can therefore be recorded directly without the use of subtraction procedures or pharmacological blockers. The charge moved by the exchange was studied by loading outer segments with a known amount of calcium and then recording the exchange current on return to a Na(+)-containing solution. Calcium is not sequestered to any significant extent in a slowly exchanging internal store, as the charge recovered is unaffected if admission of the Na(+)-containing solution is delayed for 40 s. The number of charges flowing into the cell in exchange for each Ca2+ ion extruded was found not to deviate significantly from one over a wide range of ionic conditions and membrane potentials. These results show that the stoichiometry of the exchange is fixed over a wide range of conditions, and that the size of the inward exchange current is therefore directly proportional to the rate of Ca2+ efflux through the carrier.


INTRODUCTION
Calcium enters the outer segment of rod photoreceptors through the light-sensitive channel (Hodgkin, McNaughton, and Nunn, 1985), and is expelled by an electrogenic exchange mechanism in which the efflux of calcium is linked to an influx of sodium (Yau and Nakatani, 1984;Nunn, 1985, 1987;McNaughton, Cervetto, and Nunn, 1986;Schnetkamp, 1986;Lagnado, Cervetto, and McNaughton, 1988). This mechanism is usually termed the Na:Ca exchange, although it has recently been demonstrated that K ÷ is cotransported with Ca 2+ in rods, so that the energy required for Ca z+ efflux is provided by both the Na + gradient and the K + gradient (Cervetto, Lagnado, Perry, Robinson, and McNaughton, 1989). In common with the exchange in cardiac muscle (Kimura, Miyamae, and Noma, 1987;Lipp and Pott, 1988;Bridge, Smolley, and Spitzer, 1990), the carrier generates an inward membrane current while extruding Ca 2+, and this property can be exploited to study the exchange using electrophysiological methods (Lagnado and McNaughton, 1990).
This paper describes experiments in which isolated rod outer segments under whole-cell voltage clamp have been used to investigate the average net charge transported by the Na:Ca,K exchange during a cycle in which a single Ca 2+ ion is extruded. Previous measurements using suction pipettes to record the membrane current have found some variability in the apparent stoichiometry, with between 0.94 and 0.74 charges flowing into the outer segment in exchange for every Ca 2+ ion extruded (Yau and Nakatani, 1984;Hodgkin et al., 1987). These observations raise the possibility that the stoichiometry of the exchange may vary depending on the conditions in which it is measured. An explicit test of this possibility is important both for understanding the mode of operation of this carrier, and for the design and interpretation of experiments that use the exchange current as a measure of the rate of Ca 2+ transport. We have carried out measurements of the net charge transported by the exchange over a range of membrane potentials and external concentrations of Na +, Ca 'z+, and K +. Although these various conditions caused the saturated exchange current to vary over a 40-fold range, there was no significant deviation from a coupling ratio of one charge entering per calcium extruded. The size of the exchange current can therefore be taken as a direct measure of the rate of Ca 2+ extrusion over a range of conditions. Preliminary reports of this work have been presented McNaughton, 1987, 1988).

Preparation
For each experiment a larval tiger salamander (Ambystoma tigrinum) was dark adapted for at least 1 h, decapitated, and pithed. The procedure for the mechanical dissociation of single rods from the retina (described by Hodgkin, McNaughton, Nunn, and Yau, 1984) also yields a large number of detached rod outer segments lacking the inner segment and the cell nucleus. The morphology of these outer segments is similar to that of outer segments of intact, functional rods. When the interior of these isolated outer segments is perfused with an appropriate internal solution through a whole-cell pipette the I-V relation of the light-sensitive current and the kinetics and sensitivity of the response to light flashes are similar to those of intact rods (Sather and Detwiler, 1987).

Electrical Recording
Whole-cell pipettes were drawn from 100 ~1 borosilicate measuring capillaries (Farmalitalia Carlo Erba, Milan, Italy) pulled in two stages on a horizontal puller (BB-CH; Mecanex, Geneva, Switzerland). The internal diameter of the tip was typically 0.7-1.0 ~m and the "bubble number" measured in methanol was 5.0-5.8 (Corey and Stevens, 1983). The pipette resistance when filled with intracellular solution was 3-10 MfL Fire polishing of the tip was not found necessary, nor were the shanks treated to reduce stray capacitance since only relatively slowly changing currents were recorded. Whole-cell recordings were made using a patch-clamp amplifier (model 8900; Dagan Corp., Minneapolis, MN) with a 10-GI~ feedback resistor in the headstage, and run at 1 kHz bandwidth. Seal resistances were between 1 and 50 GIL and the access resistance (which was not compensated) was typically 10-40 MfL The voltage drop across a resistance of 40 Mfl will be < 4 mV with even the largest exchange currents recorded in the present study (for example, see Fig. 5). Data were stored on magnetic tape and digitized for subsequent analysis using a PDP 11-73 minicomputer. Digital smoothing was performed in some cases by convolution with a Gaussian filter; the SD of the filter is given where appropriate in figure legends.
For mechanical stability the isolated outer segment was held end-on against the mouth of a suction pipette (internal tip diameter typically 7-9 Ixm). The suction pipette was filled with a Ringer solution containing 110 mM Li + and nominally zero Ca 2+ (with no added EGTA) to prevent activation of the Na:Ca,K exchange, in either the forward or the backwards mode, in the area of membrane within the suction pipette mouth.

Soluti.ons
Solutions bathing the outer segment were changed using the multi-pipe system described by Hodgkin et al. (1985), except that the pneumatic actuator was replaced by a stepping motor designed by the late Dr. Brian Nunn (see Hodgkin et al., 1987). Solution changes were typically complete in 50 ms.
The composition of normal Ringer was (in mM): 110 NaC1, 2.5 KCI, 1.6 MgC12, 1 CaCI~, and 10 HEPES, neutralized to pH 7.6 with tetramethylammonium hydroxide (6 mM). For Ca~÷-free or divalent-free solutions the CaCI~ and MgCI 2 were omitted and 1 mM EGTA or 1 mM EDTA was added as appropriate. Low-Na + solutions were made by replacement of NaC1 with LiCI, which does not activate the exchange (Hodgkin and Nunn, 1987). The high-Na + solution used to cause close-to-maximal activation of the exchange (see Fig. 5) contained 220 mM NaCI and no other cations; this solution was hypertonic and caused the outer segment to shrink noticeably, although the effects were fully reversible and caused no appreciable change in the light response recorded after returning to normal Ringer. This level of hypertonicity did not appear to affect the properties of the Na:Ca,K exchange because in a control experiment identical activation of the exchange was observed in an isotonic solution containing 110 mM NaCI and in a hypertonic solution containing 110 mM NaCI plus 110 mM LiC1.
The isotonic CaC12 solution used for introducing a Ca ~÷ load into the outer segment contained (in mM): 77.5 CaCI 2, 0.5 3-isobutyl-l-methylxanthine (IBMX), and 10 HEPES, pH 7.6. The phosphodiesterase inhibitor IBMX was included to keep open the light-sensitive channels, which would otherwise quickly close when the substantial rise in intracellular calcium inhibits the guanylate cyclase (Lolley and Racz, 1982;Hodgkin et al., 1985;Koch and Stryer, 1988).
The filling solution for the whole-cell pipette was Na+-free, containing (in mM): 110 K-aspartate, 3 MgC12, 1 ILzATP, 1 Li~GTP, 0.06 3'-5'-cyclic GMP (cGMP), 0.02 bis(2aminophenoxy) ethane-N,N,N',N'-tetraacetate (BAPTA), and 10 PIPES, neutralized to pH 7.2 with KOH. In some experiments the cGMP was omitted, which reduced the size of the light-sensitive current but otherwise had no effect on the results reported. In later experiments the 20 p,M of the calcium buffer BAPTA was also omitted, with no significant effect on the exchange currents.

Electrical Characteristics of Isolated Outer Segments
An example of a typical whole-cell recording from an isolated rod outer segment is shown in Fig. 1 A. Immediately after obtaining access to the cell interior the membrane current recorded under voltage clamp was zero, but an inward light- sensitive current gradually developed and increased to normal levels. The initial lack of any light-sensitive current in the isolated outer segment suggests that it is depleted of cGMP, the internal transmitter that opens light-sensitive channels (Fesenko, Kolesnikov, and Lyubarski, 1985). The activation of a light-sensitive current in the absence of cGMP in the patch pipette shows that the outer segment possesses an endogenous cyclase to convert GTP to cGMP (see also Sather and Detwiler, 1987). Fig. 1 B shows that the presence of 60 I~M cGMP in the patch solution accelerates the rate at which the light-sensitive current develops in an isolated outer segment (note the shorter time-scale relative to that in Fig. 1 A). In this example the time to half-activation of the light-sensitive current (t~/2) was ~ 6.5 s, whereas in Fig. 1 A, in the absence of exogenous cGMP, it was ~ 85 s. In 35 experiments in which 60 v~M cGMP was present in the patch pipette, t~/2 was 11.5 -+ 1.5 s, and in 26 experiments in which cGMP was absent, tl/z was 51.2 -+ 6.0 sec. There was no systematic difference in the pipette access resistance in these two conditions, so it appears that the rate at which cGMP diffuses into the outer segment is considerably greater than the rate at which it can be synthesized by the endogenous guanylate cyclase. This may be because the outer segment is initially loaded with Ca ~+, which is known to inhibit the cyclase (Lolley and Racz, 1982;Koch and Stryer, 1988).
The mean amplitude of the light-sensitive current with 1 mM ATP and 1 mM GTP in the patch solution was 35 + 3 pA (mean + SEM, n = 18) in the 5-15-min period after obtaining a whole-cell recording with the membrane clamped at -44 mV. In the additional presence of 60 wM cGMP the current at -44 mV was 88 -10 pA (n = 10). This concentration of cGMP was included in the patch solution in the majority of the experiments to be described, because as well as increasing the light-sensitive current and therefore facilitating the Ca 2+ loading of the outer segment, exogenous cGMP appeared to prolong cell survival and to counteract the gradual decrease in the light-sensitive current normally observed during a whole-cell recording. Fig. 1 shows that there is negligible current flow at the plateau of a saturating light response, reflecting the high input resistance of the isolated outer segment when the light-sensitive channels are shut (Baylor and Lamb, 1982;Baylor and Nunn, 1986). In keeping with this idea, a 30-mV hyperpolarization ( Fig. 1 B) caused a negligible shift in the current measured at the plateau of the light response. The apparent input resistances in the light of the outer segments shown in Fig. 1, A and B, were typical, being 10 and 12 GI~, respectively. It is likely that the input resistance of the outer segment in bright light is considerably higher than these figures and that the major contribution to the apparent input resistance results from the imperfect seal between the membrane and the glass of the patch pipette. An upper limit to the lightinsensitive conductance of the outer segment can be obtained from the highest input resistance measured in bright light, which was 45 G~, corresponding to a conductance of 22 pS, or less than the conductance of many single channels. This estimate is similar to that obtained by Baylor and Nunn (1986), and supports the idea that the light-sensitive channel is the only ionic channel open in the outer segment membrane under normal conditions.

Isolation of the Na:Ca, K Exchange Current
The method used to introduce a known Ca 2+ load into the outer segment and measure the Na:Ca,K exchange current is similar to that adopted by Hodgkin et al. (1987) (Fig. 2 A )  to a normal Ringer solution, activating an inward current caused by the electrogenic exchange of external Na + for internal Ca 2+ (Yau and Nakatani, 1984;Hodgkin et al., 1987;; reviewed by Lagnado and McNaughton, 1989). The exchange current remained saturated at -19 pA for ~9 s in this experiment, and then declined to zero as the Ca ~÷ load was removed. Note that the membrane current in bright light in both isotonic calcium solution and Ringer are equal to zero; because of the high resistance of the seal between the whole-cell pipette and the outer segment, no subtraction procedure is necessary to remove junction currents caused by the different mobilities of the ions in the two solutions. The mean value of the saturated exchange current was 12.5 -1.3 pA in normal Ringer solution at a membrane potential of -14 mV (Table I), which is close to the value of 20 pA measured in intact rods with a suction pipette (Hodgkin et al., 1987). Part of the difference can be explained by the dependence of exchange current on membrane potential, which would have been about -55 mV for an intact rod in bright light (Baylor and Nunn, 1986).

Estimating the Net Charge Movement
The net charge moved by the Na:Ca,K exchange during the extrusion of a single Ca 2+ ion, q', was obtained as shown in Fig. 2 B, using the assumption that all of a Ca 2+ load introduced into the outer segment is removed by the activity of the Na:Ca,K exchange (see below). The amount of Ca ~+ introduced into the outer segment during the exposure to isotonic CaCI~ was calculated by integrating the light-sensitive current. The total charge (QJoJ in this exposure was 512 pC, equivalent to 1.6 x 109 Ca ~+ ions in a cytoplasmic volume of 1 pl. The total charge entering the outer segment during the activation of the exchange (Q~xch), measured by integrating the exchange current, was 245 pC. The exchange must remove both the resting Ca 2+ load present in the outer segment in darkness at the start of the experiment and the incremental load introduced by exposure to isotonic CaCI 2. The component of Q~xch attributable to extrusion of the resting Ca 2+ load can be calculated from the integral of the exchange current observed after delivering a saturating flash in Ringer (see, for instance, Fig. 3 of Hodgkin et al., 1987) and in this example was 2 pC; the corrected value of Q~xch, attributable to the charge transferred due to the removal of the Ca 2+ load, was therefore 243 pC. The charge moved per calcium ion extruded, q ', is equal to 2Q~xch/QJo~d, and is therefore 0.95 in this experiment. The mean value of q' under these conditions (Ringer at -14 mV) was 0.98 -+ 0.03 (see Table II).

Assumptions of the Method Used for Measuring Exchange Stoichiometry
The validity of the method used to estimate q' depends on several assumptions (see below), one of the most important being that all of the Ca 2+ load is removed by the Na:Ca,K exchange. The possibility of Ca 2+ sequestration within the outer segment, or of Ca 2+ removal by a Na+-independent extrusion process, was tested by the type of experiment shown in Fig. 3. A Ca 2÷ load was introduced into an outer segment using a protocol similar to that in Fig. 2, but after shutting off the Ca 2+ influx the outer segment was transferred to a solution containing 110 mM Li +, 0 Ca 2+. The extrusion of the Ca 2+ load through the Na:Ca,K exchange was then activated by returning to normal Ringer solution. The value of q' obtained in this experiment was 1.04, which is not significantly different from the value of unity found when the load was extruded immediately (see Table II). We conclude that the calcium load remains within the outer segment in the absence of external Na +, and is neither sequestered into an inaccessible store nor extruded from the outer segment by a Nao-independent mechanism, nor does calcium diffuse to any appreciable extent into the whole-cell pipette. Similar results were obtained in three other cells, in which the exchange was disabled for 20-40 s after the introduction of the Ca 2+ load, and no difference was observed when using isotonic CaC12 rather than isotonic LiCI 2 to disable Ca '+ effiux through the exchange. We conclude that in the conditions of our experiments the time constant of Ca 2+ loss through mechanisms other than the Na:Ca,K exchange is > 100 s. The method of estimating the net charge movement through the Na:Ca,K exchange depends in addition on the following assumptions: The current in isotonic CaCl2 is carried only by Ca 2+ ions. The principal cation in the patch pipette solution is K +, which has been shown to pass through the light-sensitive channel (Capovilla, Cervetto, and Torre, 1983;Hodgkin et al., 1985). The outward K + flux is, however, likely to be negligible because the light-sensitive channel appears to behave as a single-file pore with a permeability to Ca 2+ at least 25 times that for monovalent cations (Hodgkin et al., 1985;Zimmerman and Baylor, 1986;Menini, Rispoli, and Torre, 1988).
There is a negligible Ca 2+ influx other than through light-sensitive channels, In some preparations with a poor seal between the whole-cell pipette and the membrane values of q' greatly in excess of one charge exchanging for one Ca 2+ were observed. Typically in such preparations a net charge transfer of close to one charge per Ca 2+ extruded was observed at the start of the experiment when the seal resistance was high, and as the seal deteriorated to 1 GI~ or less the apparent stoichiometry changed in a direction consistent with an increasing electrically silent entry of Ca 2+ (presumably as CaC12) during the exposure to isotonic calcium solution. In contrast, higher seal resistances (3-45 GI~) gave consistent values of the exchange stoichiometry, and so to give a margin of safety a lower limit of 5 G~ was set on the acceptable seal resistance. About 50 measurements made when the seal resistance was below 5 G~ were rejected.
There is no Ca 2+ e~ux during the loading period. The absence of Na + in the external solution will prevent any Ca 2+ e~]ux through forward Na:Ca,K exchange. The possibility of Ca 2÷ effiux through an ATP-driven pump cannot be discounted, but will make a negligible contribution in the conditions of the present experiments, as it is estimated that the maximum Ca 2+ current through such a pump is < 0.4 pA (McNaughton et al., 1986), so that the maximum efflux during a typical 5-s exposure is 2 pC compared with a typical load of 300--400 pC. Further evidence against a significant Na+-independent transport across the outer segment membrane is presented above (Fig. 3). There is negligible Ca 2+ influx through reversed Na:Ca,K exchange during loading.

Ringer
The strongest evidence against this possibility is provided by the demonstration that Ca 2+ influx through the exchange depends absolutely on the presence of external K + (Cervetto et al., 1989). K + was not included in the isotonic CaClz solution, thus blocking reversal of the exchange.   Fig. 4 A compares the exchange currents recorded at external Na ÷ concentrations of 110 and 75 raM. The reduction in [Na+]o reduces the saturated exchange current by 50% and slows the removal of the Ca 2÷ load (see also Hodgkin and Nunn, 1987). In this example, q' in 75 mM [Na+]o was 1.02, and the mean value in this solution was 1.04 -+ 0.05 (five experiments in four cells). An experiment using a wider range of [Na+]o is shown in Fig. 5. In this experiment the exchange current was changed over a 7.2-fold range without a significant deviation from unity in the value of charge transported per calcium ion extruded. The reduction in the magnitude of the saturated exchange current in reduced [Na+]o is therefore due to a reduction in the rate of operation of the carrier rather than to a change in the stoichiometry of the exchange.  6 mM) are collected together as these ions did not appear to alter the stoichiometry of the exchange. The third column gives the number of observations in each condition, and the fourth column gives the number of different outer segments in which these measurements were made.

The Net Charge Movement under Various Conditions
The thin trace in Fig. 4 B shows an experiment in which the effect of membrane potential on the exchange stoichiometry was examined. A 50-mV hyperpolarization increased the saturated exchange current by 47% and accelerated the removal of the Ca 2+ load, but the value of q' at -64 mV was unchanged (0.99 in this experiment) and the mean value for this hyperpolarization was 1.00 + 0.05 (see Table II).
The results from these experiments are shown in Fig. 6. The net charge movement per calcium ion transported is given as a function of [Na+]o at a membrane potential of -14 mV (filled circles), and at Vm = -64 mV (filled squares). Varying [Na+],, between 35 and 220 mM does not cause the value of q' to differ significantly from unity. Over the range of conditions investigated here, therefore, there is no effect either of the electrochemical gradient for Na + or of the membrane potential on the stoichiometry of the exchange. The exchange current in these experiments varied over a 40-fold range, from 2 pA in 35 mM Na +, 1.6 mM Mg ~+, and 2.5 mM K +, to >80 pA in 220 mM Na +, 0 Mg 2+, and 0 K +. The values of q' measured under a wider variety of conditions are shown in Table  II The net charge movement through the Na:Ca,K exchange under a variety of conditions. Each point is the value ofq' at the external [Na ÷] indicated, either at -14 mV (@) or -64 mV (11). All the results shown were obtained in solutions containing 0 Ca 2+, but the results in the presence and absence of external K ÷ (2.5 raM) and Mg 2+ (1.6 raM) are collected together as these ions did not appear to affect the value ofq'. Bars show -+SEM. Over the range of conditions tested the value of q' did not differ significantly from 1. membrane potential (-64 to + 16 mV), and of the presence or absence of external Ca 2+ (1 mM), K + (2.5 mM), or Mg ~+ (1.6 mM). From the collected results, the net charge movement through the exchange during the extrusion of a single Ca 2+ ion was calculated to be q' = 1.004 +-0.011 (84 measurements on 16 cells).

DISCUSSION
The main conclusion from these experiments is that the Na:Ca,K exchange in rods transports one net positive charge into the cell during the extrusion of a single Ca 2+ ion, and that the charge transfer is independent of the electrochemical gradients for Na ÷, Ca 2+, and K ÷ over a wide range. The charge transfer is also independent of whether the exchange is operating in the forward or the reversed mode (Cervetto et al., 1989). These properties simplify the interpretation of kinetic studies in which the exchange current is measured at different membrane potentials and/or ionic conditions ; the size of the exchange current can be taken as a direct measure of the rate of Ca 2+ transfer through the carrier.
The charge moved by the Na:Ca,K exchange in exchange for each Ca z÷ extruded has been found using the suction pipette method to be 0.94 in the absence of [Ca~+]o by Yau and Nakatani (1984) and 0.74 in normal Ringer by Hodgkin et al. (1987). The deviations from a value of unity are probably related to the disadvantages inherent in the suction pipette method, many of which are eliminated by the more direct technique used in this paper. First, the membrane potential is not under experimental control in the suction pipette method. Second, the suction pipette typically collects only ~50% of the outer segment membrane current, and the possibility remains open that this fraction might change in the course of an experimental manipulation. Third, the seal resistance of the whole-cell pipette (typically 10-20 GI~) is three orders of magnitude higher than that of the suction pipette (5-10 Mr/). The currents caused by junction potentials between solutions of different ionic composition, which must be removed from suction pipette records by subtraction, are therefore typically too small in the whole-cell records to require subtraction. Finally, the internal ionic environment is under experimental control when using the whole-cell technique, an advantage which has been used in the present study to remove internal Na ÷.
The simplest stoichiometry consistent with the influx of a single charge during the efflux of a single Ca 2÷ ion is one in which 3Na + ions exchange with 1Ca z+, as was found in recent experiments investigating the reversal potential of the exchange in cardiac sarcolemmal vesicles (Reeves and Hale, 1984) and intact ventricular myocytes (Ehara, Matsuoka, and Noma, 1989). In rod outer segments, though, Cervetto et al. (1989) have demonstrated that the reversed exchange, mediating Ca 2+ influx, depends on the presence of external K +, and by varying [Na+]o, [Ca2+]o , [K+]o, and membrane potential, it was found that the equilibrium condition for the exchange was best described by the relation expected for the transport of 4Na ÷ ions in exchange for 1K + and 1Ca 2÷. Evidence for Na:Ca,K cotransport in photoreceptors is also provided by the results of flux measurements in bovine rod outer segments, where it has been found that external K stimulates Ca uptake via the reversed exchange, and internal K stimulates the component of Ca efflux which is dependent on external Na (Schnetkamp, Szerencsei, and Basu, 1989).
It should be pointed out, however, that the net charge movement per Ca 2+ ion transported is an average measurement over a large population of exchange molecules over many seconds. If two conditions are met the macroscopic charge flow can be equated to the charge transported per exchange cycle. First, there must be a homogenous population of exchange molecules in the outer segment membrane, and second, each cycle extruding a Ca 2÷ ion must be identical. We do not think it likely that there is more than one type of Na:Ca,K exchange, but one might imagine, for example, that half of the exchange cycles operate with a stoichiometry of 4Na+: 1Ca ~+ (net charge +2) and that the other half operate with a stoichiometry of 4Na+: ICa2+,2K + (net charge 0), leading to an average net movement of one positive charge per Ca + ion. These two cycles would have to be tightly coupled since we find that the exchange is strictly dependent on K +. Such possibilities raise the issue of the ways in which the microscopic behavior of the exchange might be studied. Clearly there is no equivalent to single channel recording at present, but if the unitary conductance of the exchange is large enough, another possibility may be the use of noise analysis.
An investigation of the net charge movement through the cardiac Na:Ca exchange has recently been carried out along similar lines by Bridge et al. (1990) using isolated ventricular myocytes under whole-cell voltage clamp. These authors used caffeine to prevent Ca 2+ uptake and release from intracellular stores, and the Ca 2+ channel blocker nifedipine to isolate the Ca ~+ current. They found the charge entering the cell through the Ca 2+ channels to be about twice that moved by the exchange in expelling the Ca 2+ load, consistent with a stoichiometry of 3Na+:lCa 2+ for the exchange in this tissue. The electrochemical gradient for Na + was not changed in these experiments, but the net charge transport was fixed over a range of Ca loads.
There is evidence to suggest that the electrogenic Na:K pump also operates with a fixed stoichiometry (Thomas, 1969). Using dialyzed squid giant axons under voltage clamp, Rakowski, Gadsby, and De Weer (1989) simultaneously identified 22Na fluxes and membrane current through the pump by specifically blocking its activity with a cardiotonic steroid. They found that the ratio of the change in 22Na efflux to the change in the membrane current on applying the steroid to be independent of membrane potential (between 0 and -60 mV) and external [Na ÷] (between 0 and 390 mM). In all conditions the results were not significantly different from those expected for a stoichiometry of 3Na+:2K +, showing that changes in the Na:K pump current simply reflect changes in the rate at which the carrier operates.
We are grateful to Luigi Cervetto, Richard Perry, and David Robinson for their comments on the manuscript. This work was supported by the MRC, by a George Henry Lewes Studentship to L. Lagnado, and by a Nuffield Science Research Fellowship to P. McNaughton.