Potassium channels in Necturus proximal tubule

Potassium channels from the apical and basolateral membranes of Necturus proximal tubule were studied using the patch-clamp technique. The conductance of the basolateral channel was dependent on the pipette K+ concentration (apparent Km, 65.5 mM K+; maximum channel conductance, 49.8 pS). The permeability ratio (PK+/PNa+) was approximately 10:1. The fractional open time increased with hyperpolarization, whereas mean open times did not change. Ba2+ (0.1 mM pipette concentration) blocked the channel and reduced the mean open time. The apical K+ channel was activated with depolarization and had a slope conductance of 60 pS in the inside-out configuration (100 mM KCl in the pipette and 2.5 mM KCl in the bath). The K+-Na+ selectivity ratio was 32:1. These different channel types will allow independent control of the apical and basolateral membrane K+ conductances.

Fluid reabsorption by Necturus proximal tubule perfused with solutions of normal and reduced osmolarity

Fluid absorption in Necturus proximal tubule was studied when the kidneys were perfused with solutions of different osmolarities. The rate of fluid absorption was inversely proportional to the perfusion fluid osmolarity, while Na uptake remained constant. No difference was detected between the collected and injected luminal fluid, i. e. reabsorption was isotonic at normal and reduced osmolarities. The transtubular osmotic permeability remained fairly constant under the different per­fusion osmolarities. Using our experimental results to test various models based on osmotic equilibration across the tubule wall we show that none of these provides an adequate mechanism for fluid absorption in this epithelium.

Dissociation of proximal tubular glucose and Na+ reabsorption by amphotericin B

The effect of amphotericin B on glucose and Na+ transport was studied in the Necturus proximal tubule and in microvillus membrane vesicles isolated from the rabbit renal cortex. In the Necturus experiments, the rate constants for disappearance of radiolabeled glucose (kG) and mannitol (kM) from the tubular lumen were determined by stop-flow microperfusion. Saturability and Na+-dependence of glucose reabsorption was confirmed, since kG was reduced by raising intratubular glucose from 1 to 5 mM or by replacing intratubular Na+ with choline. Neither maneuver affected kM. Intratubular amphotericin B (10 microgram/ml), previously shown to stimulate active Na+ reabsorption in the Necturus proximal tubule, inhibited kG with no effect on kM. In the membrane vesicle preparation, amphotericin inhibited the uphill glucose uptake which results from imposing a NaCl gradient from outside to inside, but had no effect on glucose uptake in either the absence of Na+ or in the presence of Na+ when there was no Na+ gradient. Amphotericin B stimulated the uptake of Na+ by the vesicles. The observed dissociation of glucose and Na+ transport by amphotericin B is consistent with the concept that proximal tubular glucose reabsorption is energized by the luminal membrane Na+ gradient and is not directly linked to active Na+ transport per se.

Luminal Na+ entry into Necturus proximal tubule cells

The dependence of intracellular Na+ activity on the electrical driving force across the luminal membrane and the presence of Cl- in the luminal perfusate was studied in Necturus proximal tubule. Intracellular Na+ and K+ activities were measured with microelectrodes filled with liquid ion exchanger. Perfusion of the tubule lumen with a NaCl-free solution caused cell Na+ activity to fall from the control value of 29.7 to 6.6 mM. In the absence of luminal driving force across the luminal membrane in accordance with simple diffusion of Na+ across this membrane. When the tubule lumen contained Na+ and Cl-, an electrically neutral component of Na+ entry into cells from the lumen appeared in addition to the diffusional component of Na+ entry.