Current-voltage relationships for the plasma membrane and its principal electrogenic pump inNeurospora crassa: I. Steady-state conditions

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Abstract
The nonlinear membrane current-voltage relationship (I–V curve) for intact hyphae ofNeurospora crassa has been determined by means of a 3-electrode voltage-clamp technique, plus “quasi-linear” cable theory. Under normal conditions of growth and respiration, the membraneI–V curve is best described as a parabolic segement convex in the direction of depolarizing current. At the average resting potential of −174 mV, the membrane conductance is ≈190 μmhos/cm2; conductance increases to ≈240 μmhos/cm2 at −300 mV, and decreases to ≈130 μmhos/cm2 at 0 mV. Irreversible membrane breakdown occurs at potentials beyond this range. Inhibition of the primary electrogenic pump inNeurospora by ATP withdrawal (with 1mm KCN) depolarizes the membrane to the range of −40 to −70 mV and reduces the slope of theI–V curve by a fixed scaling factor of approximately 0.8. For wild-typeNeurospora, compared under control conditions and during steady-state inhibition by cyanide, theI–V difference curve — presumed to define the current-voltage curve for the electrogenic pump — is a saturation function with maximal current of ≈20 μA/cm2, a half-saturation potential near −300 mV, and a projected reversal potential of ca. −400 mV. This value is close to the maximal free energy available to the pump from ATP hydrolysis, so that pump stoichiometry must be close to 1 H+ extruded:1 ATP split. The time-courses of change in membrane potential and resistance with cyanide are compatible with the steady-stateI–V curves, under the assumption that cyanide has no major effects other than ATP withdrawal. Other inhibitors, uncouplers, and lowered temperature all have more complicated effects. The detailed temporal analysis of voltage-clamp data showed three time-constants in the clamping currents: one of 10 msec, for charging the membrane capacitance (0.9 μF/cm2) a second of 50–75 msec; and a third of 20–30 sec, perhaps representing changes of intracellular composition.