External potassium (K) activates the inward rectifier ROMK (K(ir)1.1) by altering the pH gating of the channel. The present study examines this link between external K and internal pH sensitivity using both the two-electrode voltage clamp and the perfused, cut-open Xenopus oocyte preparation. Elevating extracellular K from 1 mM to 10 mM to 100 mM activated ROMK channels by shifting their apparent pK(a) from 7.2 +/- 0.1 (n = 6) in 1 mM K, to 6.9 +/- 0.02 (n = 5) in 10 mM K, and to 6.6 +/- 0.03 (n = 5) in 100 mM K. At any given internal pH, the number of active ROMK channels is a saturating function of external [K]. Extracellular Cs (which blocks almost all inward K current) also stimulated outward ROMK conductance (at constant 1 mM external K) by shifting the apparent pK(a) of ROMK from 7.2 +/- 0.1 (n = 6) in 1 mM K to 6.8 +/- 0.01 (n = 4) in 1 mM K + 104 mM Cs. Surprisingly, the binding and washout of the specific blocker, Tertiapin-Q, also activated ROMK in 1 mM K and caused a comparable shift in apparent pK(a). These results are interpreted in terms of both a three-state kinetic model and a two-gate structural model that is based on results with KcsA in which the selectivity filter can assume either a high or low K conformation. In this context, external K, Cs, and Tertiapin-Q activate ROMK by destabilizing the low-K (collapsed) configuration of the selectivity filter.

A ouabain sensitive inward current occurs in Xenopus oocytes in Na + and K + -free solutions. Several laboratories have investigated the properties of this current and suggested that acidic extracellular pH (pH o ) produces a conducting pathway through the Na + /K + pump that is permeable to H + and blocked by [Na + ] o . An alternative suggestion is that the current is mediated by an electrogenic H + -ATPase. Here we investigate the effect of pH o and [Na + ] o on both transient and steady-state ouabainsensitive current. At alkaline or neutral pH o the relaxation rate of pre-steady-state current is an exponential function of voltage. Its U-shaped voltage dependence becomes apparent at acidic pH o , as predicted by a model in which protonation of the Na + /K + pump reduces the energy barrier between the internal solution and the Na + occluded state. The model also predicts that acidic pH o increases steady-state current leak through the pump. The apparent pK of the titratable group(s) is 6, suggesting that histidine is involved in induction of the conductance pathway. 22 Na efflux experiments in squid giant axon and current measurements in oocytes at acidic pH o suggest that both Na + and H + are permeant. The acid-induced inward current is reduced by high [Na + ] o , consistent with block by Na + . A least squares analysis predicts that H + is four orders of magnitude more permeant than Na + , and that block occurs when 3 Na + ions occupy a low affinity binding site (K 0.5 = 130 ± 30 mM) with a dielectric coeffcient of 0.23 ± 0.03. These data support the conclusion that the ouabain-sensitive conducting pathway is a result of passive leak of both Na + and H + through the Na + /K + pump.

Short (<1 sec) duration depolarization of Xenopus laevis oocytes to voltages greater than +40 mV activates a sodium-selective channel (Na(x)) with sodium permeability five to six times greater than the permeability of other monovalent cations examined, including K+, Rb+, Cs+, TMA+, and Choline+. The permeability to Li+ is about equal to that of Na+. This channel was present in all oocytes examined. The kinetics, voltage dependence and pharmacology of Na(x)distinguish it from TTX-sensitive or epithelial sodium channels. It is also different from the sodium channel of Xenopus oocytes activated by prolonged depolarization, which is more highly selective for Na+, requires prolonged depolarization to be activated, and is blocked by Li+. Intracellular Mg2+ reversibly inhibits Na(x), whereas extracellular Mg2+ does not have an inhibitory effect. Intracellular Mg2+ inhibition of Na(x), is voltage dependent, suggesting that Mg2+ binding occurs within the membrane field. Eosin is also a reversible voltage-dependent intracellular inhibitor of Na(x), suggesting that a P-type ATPase may mediate the current. An additional cytoplasmic factor is involved in maintaining Na(x) since the current runs down in internally perfused oocytes and excised membrane patches. The rundown is reversible by reintroduction of the membrane patch into oocyte cytoplasm. The cytoplasmic factor is not ATP, because ATP has no effect on Na(x) current magnitude in either cut-open or inside-out patch preparations. Extracellular Gd3+ is also an inhibitor of Na(x). Na(x) activation follows a sigmoid time course. Its half-maximal activation potential is +100 mV and the effective valence estimated from the steepness of conductance activation is 1.0. Na(x) deactivates monoexponentially upon return to the holding potential (-40 mV). The deactivation rate is voltage dependent, increasing at more negative membrane potentials.

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