Voltage dependence of Na/K pump current inXenopus oocytes

Abstract

Stage V and VI (Dumont, J.N., 1972.J. Morphol. 136:153–180) oocytes ofXenopus laevis were treated with collagenase to remove follicular cells and were placed in K-free solution for 2 to 4 days to elevate internal [Na]. Na/K pump activity was studied by restoring the eggs to normal 3mm K Barth's solution and measuring membrane current-voltage (I–V) relationships before and after the addition of 10 μm dihydroouabain (DHO) using a two-microelectrode voltage clamp. Two pulse protocols were used to measure membraneI–V relationships, both allowing membrane currents to be determined twice at each of a series of membrane potentials: (i) a down-up-down sequence of 5 mV, 1-sec stair steps and (ii) a similar sequence of 1-sec voltage pulses but with consecutive pulses separated by 4-sec recovery periods at the holding potential (−40 mV). The resulting membraneI−V relationships determined both before and during exposure to DHO showed significant hysteresis between the first and second current measurements at each voltage. DHO difference curves also usually showed hysteresis indicating that DHO caused a change in a component of current that varied with time. Since, by definition, the steady-state Na/K pumpI−V relationship must be free of hysteresis, the presence of hysteresis in DHO differenceI−V curves can be used as a criterion for excluding such data from consideration as a valid measure of the Na/K pumpI−V relationship. DHO differenceI−V relationships that did not show hysteresis were sigmoid functions of membrane potential when measured in normal (90mm) external Na solution. The Na/K pump current magnitude saturated near 0 mV at a value of 1.0–1.5 μA cm⁻², without evidence of negative slope conductance for potentials up to +55 mV. The Na/K pump current magnitude in Na-free external solution was approximately voltage independent. Since these forward-going Na/K pumpI−V relationships do not show a region of negative slope over the voltage range −110 to +55 mV, it is not necessary to postulate the existence of more than one voltage-dependent step in the reaction cycle of the forward-going Na/K pump.