Models for coupling of salt and water transport are developed with two important assumptions appropriate for leaky epithelia. (a) The tight junction is permeable to both sale and water. (b) Active Na transport into the lateral speces is assumed to occur uniformly along the length of the channel. The proposed models deal specifically with the intraepithelial mechanism of proximal tubular resbsorption in the Necturus kidney although they have implications for epithelial transport in the gallbladder and small intestine as well. The first model (continuous version) is similar to the standing gradient model devised by Diamond and Bossert but used different boundary conditions. In contrast to Diamond and Bossert's model, the predicted concentration profiles are relatively flat with no sizable gradients along the interspace. The second model (compartment version) expands Curran's model of epithelial salt and water transport by including additional compartments and considering both electrical and chemical driving forces for individual Na and Cl ions as well as hydraulic and osmotic driving forces for water. In both models, ion and water fluxes are investigated as a function of the transport parameters. The behavior of the models is consistent with previously suggested mechanisms for the control of net transport, particularly during saline diuresis. Under all conditions the predicted ratio of net solute to solvent flux, or emergent concentration, deviates from exact isotonicity (except when the basement membrane has an appreciable salt reflection coefficient). However, the degree of hypertonicity may be small enough to be experimentally indistinguishable from isotonic transport.

The role of K+ channels in cell osmoregulation was investigated by using the patch-clamp technique. In cell-attached patches from Necturus proximal tubule, the short-open-time K+ channel at the basolateral membrane could be stretch-activated by pipette suction, where a negative pressure of 6 cm H2O (588.6 Pa) was sufficient to increase the open probability of the channel by a factor of 4.0 +/- 0.8 (n = 7 tubules). A 50% reduction in bath osmolarity increased cell volume by 66 +/- 10% and increased the K+-channel open probability by a factor of 5.8 +/- 1.4 (n = 7) in the same cell-attached patches that were activated by pipette suction. A kinetic analysis indicates one open state and at least two closed states for this epithelial K+ channel. Both suction and swelling shorten the longest time constant of the closed-time distribution by a factor of 3, without significant effect on either the mean open time or the shorter closed-state time constant. The similar effect of suction and swelling is consistent with the hypothesis that stretch-activated K+ channels mediate the increase in macroscopic K+ conductance that occurs during osmoregulation of amphibian proximal tubules. Calculations based on a simple model indicate that small increments in cell volume could produce statistically significant increases in K+-channel activity.

High-conductance (maxi) K channels in the apical membrane of rat and rabbit cortical collecting tubules (CCT) were studied using the patch-clamp technique. Principal cells (PC) and intercalated cells (IC) were distinguished with Hoffman modulation optics in split-open tubules. IC were further identified by staining tubules with the fluorescent mitochondrial dye, rhodamine 123. Maxi-K channels were distinguished by their high conductance (greater than 80 pS) and voltage-dependent kinetics. In CCT of rats on a low-Na diet, maxi K channels were observed in 11% of the cell-attached patches on PC and 79% of patches on IC. In rats on a normal diet, the channels were seen in 23 and 79% of patches on PC and IC, respectively. In the rabbit CCT, maxi K channels were observed in 12% (4 of 32) of the patches on PC and 82% (122 of 148) of the patches on IC. The greater abundance of channels in IC was confirmed in rat CCT using the whole-cell clamp technique. Current through the maxi K channels (IK) was measured as the tetraethylammonium (TEA)-sensitive (2.5 mM) outward current in cells equilibrated with 115 mM K and 10(-5) M Ca2+ in the pipette solution. When the cell was clamped to an internal potential of +40 mV, the average IK per cell was -4 +/- 5 pA in PC and 290 +/- 90 pA in IC. Lowering cytoplasmic Ca2+ from 10(-5) M to 10(-7) M reduced IK to 32 +/- 21 pA. Neither single Na channels nor amiloride-sensitive whole-cell currents were seen in IC. Finally, maxi K channels could be activated by pipette suction (10-40 cm H2O) in either cell-attached or inside-out patches on IC from rabbit CCT. This mechanosensitivity was observed even after chelation of free Ca2+ with ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in the pipette or the bath solutions, implying that stretch activation of these channels was not mediated by increased Ca2+ entry into the cell. The IC maxi K channel may play a role in cell volume regulation or in K secretion during elevation of luminal hydrostatic pressure.

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

ROMK channels play a key role in overall K balance by controlling K secretion across the apical membrane of mammalian cortical collecting tubule. In contrast to the family of strong inward rectifiers (IRKs), ROMK channels are markedly sensitive to intracellular pH. Using Xenopus oocytes, we have confirmed this pH sensitivity at both the single-channel and whole cell level. Reduction of oocyte pH from 6.8 to 6.4 (using a permeant acetate buffer) reduced channel open probability from 0.76 ± 0.02 to near zero ( n = 8), without altering single-channel conductance. This was due to the appearance of a long-lived closed state at low internal pH. We have confirmed that a lysine residue (K61 on ROMK2; K80 on ROMK1), NH₂ terminal to the first putative transmembrane segment (M1), is primarily responsible for conferring a steep pH sensitivity to ROMK (B. Fakler, J. Schultz, J. Yang, U. Schulte, U. Bråandle, H. P. Zenner, L. Y. Jan, and J. P. Ruppersberg. EMBO J. 15: 4093–4099, 1996). However, the apparent p K _a of ROMK also depends on another residue in a highly conserved, mildly hydrophobic area: T51 on ROMK2 (T70 on ROMK1). Replacing this neutral threonine (T51) with a negatively charged glutamate shifted the apparent p K _a for inward conductance from 6.5 ± 0.01 ( n = 8, wild type) to 7.0 ± 0.02 ( n = 5, T51E). On the other hand, replacing T51 with a positively charged lysine shifted the apparent p K _a in the opposite direction, from 6.5 ± 0.01 ( n = 8, wild type) to 6.0 ± 0.02 ( n = 9, T51K). The opposite effects of the glutamate and lysine substitutions at position 51 (ROMK2) are consistent with a model in which T51 is physically close to K61 and alters either the local pH or the apparent p K _a via an electrostatic mechanism. In addition to its effects on pH sensitivity, the mutation T51E also decreased single-channel conductance from 34.0 ± 1.0 pS ( n = 8, wild type) to 17.4 ± 1 pS ( n = 9, T51E), reversed the voltage gating of the channel, and significantly increased open-channel noise. These effects on single-channel currents suggest that the T51 residue, located in a mildly hydrophobic area of ROMK2, also interacts with the hydrophobic region of the permeation pathway.