Current-voltage relationships and voltage sensitivity of the Cl− pump inHalicystis parvula

Abstract

Summary. The current-voltage (I-V) relationships for internally perfused and nonperfused cells of Halieystis parvula were determined. In both types of cells the I-V curve shows a conspicuous region of negative slope, beginning at vacuole potentials around -30 mV and continuing to values of +20 to + 40inV. The negative slope in perfused cells is abolished by the metabolic inhibitors, darkness and low temperature. In order to determine the origin of this negative slope, we measured the voltage sensitivity of the unidirectional fluxes of CI-, Na + and K + in perfused cells. The results show that the C1-influx, which is mediated primarily by a C1-pump, increases as the vacuole potential is clamped at increasingly more negative values up to -50mV, while the other fluxes measured changed in the directions predicted by the change in electrical driving force. The voltage sensitivity of the C1-pump quantitatively accounts for the negative slope of the I-V curve. Also, we observed a large transient outward current of 10-20-sec duration following an abrupt depolarization by voltage clamping. This transient current was reduced or abolished by low temperature, which suggests that it may be due to the voltage-sensitive C1-pump. Finally, we found an inverse relationship between the transprotoplasm resistance (Rm) and the PD under standard conditions, which suggests that the activity of the electrogenic C1-pump lowers Rm, i.e., it is a conductive pump.An electrogenic pump is an active transport system which transports net ionic current and thus generates an electrical potential difference (PD) across a membrane. In separate theoretical models Finkelstein (1964) and Rapoport (1970) proposed that an electrogenic pump may be voltage sensitive and consequently contribute to the membrane conductance. There is experimental evidence for the existence of voltagesensitive, electrogenic pumps in the marine alga, Acetabularia (Gradmann, 1975), the freshwater alga, Nitella (Spanswick, 1972), a molluscan nerve (Kostyuk, Krishtal & Pidoplichko, 1972) & Curran, 1973). In the frog skin direct flux measurements have been made to support this hypothesis, but the direction of voltage sensitivity and the anatomical complexity of the skin make the interpretation equivocal.In the marine alga, Halicystis parvula, the inwardly directed C1-pump appears to be strongly electrogenic, accounting for nearly all of the -50 to -60 mV potential difference (PD) in internally perfused cells (Graves & Gutknecht, 1977). When connected to a voltage clamp device, the perfused cells of H. parvula allow the investigation of both the current-voltage relationships and the voltage dependence of the unidirectional fluxes of various ions. The existence of a negative slope region in the I-V curve of H. parvula suggested that the electrogenic C1-pump might be voltage sensitive, and we have investigated this possibility with direct determination of the voltage-dependent changes in direction and magnitude of the fluxes of CI-, Na + and K +. In addition, the effects of metab...

Chloride transport and the membrane potential in the marine alga,Halicystis parvula

“…This theoretical value of lip compares favorably with the mean of all the uninhibited PD's presented in this paper, which is -55.2 mV, and this agreement suggests that the estimated value ofR m in the light is reasonable. Furthermore, Graves and Gutknecht (1977) obtain a similar value for R m in the light by considering only the voltage sensitivity of the CI-efflux in perfused cells. Thus, both experimental and theoretical evidence support the hypothesis that an electrogenic CI-pump is responsible for virtually all of the PD in perfused cells of H. parvula.…”

“…Therefore, even though darkness causes the measured resistance to increase from about 700 to 2400 f~. cm 2 (Graves & Gutknecht, 1977), the degree to which this reflects a change in the resistance to passive ionic flow is not known. One way of indirectly estimating the effect of darkness on passive ionic flow is to measure the effect of darkness on the CI-efflux.…”

“…However, this calculation is complicated by the probability that the C1-pump contributes to the membrane conductance. For example, using 700f~-cm 2 for R,, in perfused cells (Graves & Gutknecht, 1977) and the mean SCC of 29 p~A 9 cm-2 (Graves & Gutknecht, 1976), we calculate that -20 mV is the upper limit for the potential produced electrogenically, whereas the inhibitor studies suggest that the electrogenic pump contributes -50 to -60 mV. The discrepancy may lie in the underestimation of the true Rm due to a contribution to the conductance by the electrogenic pump.…”

“…However, interpretation of the resistance data in Halicystis is complicated by the fact that the C1-pump is voltage sensitive and thus contributes directly to the membrane conductance (Graves & Gutknecht, 1977). Therefore, even though darkness causes the measured resistance to increase from about 700 to 2400 f~.…”

“…One way of indirectly estimating the effect of darkness on passive ionic flow is to measure the effect of darkness on the CI-efflux. The CI-efflux in perfused cells is the dominant dissipative flux and is highly sensitive to the electrochemical driving force (Graves & Gutknecht, 1976, 1977, which suggests it is largely due to simple diffusion. Figure 5 shows the effect of darkness on the CI-efflux from a group of six intact cells.…”