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Current-voltage relationships for the plasma membrane and its principal electrogenic pump in Neurospora crassa: I.
Abstract:
The nonlinear membrane current-voltage relationship (I-V curve) for intact hyphae of Neurospora 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 membrane I-V curve is best described as a parabolic segment convex in the direction of depolarizing current. At the average resting potential of - 174 mV, the membrane conductance is approximately 190 micronhos/cm2; conductance increase to approximately 240 micronhos/cm2 at -300 mV, and decreases to approximately 130 micronhos/cm2 at 0 mV. Irreversible membrane breakdown occurs at potentials beyond this range. Inhibition of the primary electrogenic pump in Neurospora by ATP withdrawal (with 1 mM KCN) depolarizes the membrane to the range of -40 to -70 mV and reduces the slope of the I-V curve by a fixed scaling factor of approximately 0.8. For wild-type Neurospora, compared under control conditions and during steady-state inhibition by cyanide, the I-V difference curve--presumed to define the current-voltage curve for the electrogenic pump--is a saturation function with maximal current of approximately 20 muA/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-state I-V curves, under the assumption the 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 muF/cm/2); a second of 50-75 msec; and a third of 20-30 sec, perhaps representing changes of intracellular composition.