Summary. Sudden respiratory blockade has been used to study rapid changes of the resting membrane potential, of intracellular adenosine 5'-triphosphate (ATP) levels, and of pyridine nucleotide reduction in Neurospora crassa. Membrane depolarization occurs with a first-order rate constant of 0.167 sec -1, following a lag period of about 4 sec, at 24 ~ (ambient temperature). This depolarization is several-fold too slow to be directly linked to electron transfer, as judged from the rate of pyridine nucleotide reduction, but has essentially the same rate constant as the decay of ATP. The latter process, however, shows no lag period after the respiratory inhibitor is introduced. Plots of membrane potential versus the intracellular ATP concentration yield saturation curves which are readily fitted by a Michaelis equation, to which is added a constant term representing the diffusion component of membrane potential. Parameters obtained from such fits indicate the maximal voltage which the pump can develop at high ATP levels to be 300 to 350 mV, with an apparent 1(1/2 of 2.0 rr~. The data strongly suggest that an electrogenic ion pump in the plasma membrane of Neurospora is fueled by ATP; comparison of the measured membrane potentials with the energy available from hydrolysis of ATP indicates that two ions could be pumped for each molecule of ATP split.Although the existence of electrogenic ion pumps was postulated about 50 years ago from extracellular studies on plant tissues and on epithelial membranes, their acceptance as bona fide physiological entities did not occur until the past 10 years. During this period proof has been evolving from two quite separate lines of research. First, in certain nerve and muscle preparations, where membrane potential and resistance can be measured with micropipette electrodes, it has been possible to demonstrate a precise quantitative correspondence between the currents (and fluxes) generated by the sodium pump and the voltage and resistance characteristics of the particular membrane [2,18,23,33,51,52]. Related experiments on plant and fungal preparations have given less complete data, but have clearly demonstrated the existence of membrane potentials which are extremely difficult to account for in terms of conventional ion-diffusion processes 21 J. Membrane Biol, 14

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.

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We have used volume-localized 'H NMR spectroscopy to detect and measure changes in medullary trimethylamines (TMAs) in the human kidney in vivo. Localized water-suppressed 'H spectra were collected from a volume of interest located within the renal medulla by using a stimulated echo-based localization scheme. The principal resonances in the medullary 'H spectrum were residual water (4.7 ppm), lipid (0.9-1.4 ppm), and TMAs (3.25 ppm). The TMA line width was 7-15 Hz before filtering, and the signal-to-noise ratio was 40:1. In four normal volunteers, 15 hr of dehydration led to a significant increase in urine osmolality and decrease in body weight and an increase in medullary TMAs. A subsequent water load [20 ml-(kg of body weight)-'] caused a transient water diuresis, a return to euvolemic body weight, and a significant reduction in medullary TMAs within 4 hr. These results suggest that TMAs may play an osmoregulatory role in the medulla of the normal human kidney.