The activity of ROMK channels in rat kidney tubule cells was assessed as tertiapin-Q (TPNQ)-sensitive current under whole cell clamp conditions. With an external K+ concentration of 5 mM and an internal K+ concentration of 140 mM and the membrane potential clamped to 0 mV, TPNQ blocked outward currents in principal cells of the cortical collecting duct (CCD) outer medullary collecting duct and connecting tubule (CNT). The apparent Ki was 5.0 nM, consistent with its interaction with ROMK. The TPNQ-sensitive current reversed at voltages close to the equilibrium potential for K+. The currents were reduced when the pipette solution contained ATP. In the CCD, the average TPNQ-sensitive outward current ( ISK) was 476 ± 48 pA/cell in control animals on a 1% KCl diet. ISK increased to 1,255 ± 140 pA when animals were maintained on a high-K (10% KCl) diet for 7 days and decreased to 314 ± 46 pA after 7 days on a low-K (0.1% KCl) diet. In the CNT, ISK was 360 ± 30 pA on control, 1,160 ± 110 on high-K, and 166 ± 16 pA on low-K diets. The results indicate that ROMK channel activity is highly regulated by dietary K in both the CCD and the CNT.

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent Km for three permeant ions: K+, Rb+, and NH4+. Compared with K+ (imax = 4.6 pA and Km = 10 mM at −100 mV), Rb+ had a lower permeability, a lower imax (1.8 pA), and a higher Km (26 mM). For NH4+, the permeability was reduced more with smaller changes in imax (3.7 pA) and Km (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na+, Li+, Mg2+, and Ca2+ with similar voltage dependence (apparent valence, 0.15–0.20). It prefers Mg2+ over Ca2+ and has a monovalent cation selectivity, based on the ability to displace Mg2+, of K+ > Li+ ∼ Na+ > Rb+ ∼ NH4+. Conversely, in the presence of Mg2+, the Km for K+ conductance was substantially increased. The ability of Mg2+ to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent Km for K+ conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.

The inwardly rectifying potassium channel (Kir), Kir4.1 mediates spatial K(+)-buffering in the CNS. In this process the channel is potentially exposed to a large range of extracellular K(+) concentrations ([K(+)]o). We found that Kir4.1 is regulated by K(+)o. Increased [K(+)]o leads to a slow (mins) increase in the whole-cell currents of Xenopus oocytes expressing Kir4.1. Conversely, removing K(+) from the bath solution results in a slow decrease of the currents. This regulation is not coupled to the pHi-sensitive gate of the channel, nor does it require the presence of K67, a residue necessary for K(+)o-dependent regulation of Kir1.1. The voltage-dependent blockers Cs(+) and Ba(2+) substitute for K(+) and prevent deactivation of the channel in the absence of K(+)o. Cs(+) blocks and regulates the channel with similar affinity, consistent with the regulatory sites being in the selectivity-filter of the channel. Although both Rb(+) and NH4(+) permeate Kir4.1, only Rb(+) is able to regulate the channel. We conclude that Kir4.1 is regulated by ions interacting with specific sites in the selectivity filter. Using a kinetic model of the permeation process we show the plausibility of the channel's sensing the extracellular ionic environment through changes in the selectivity occupancy pattern, and that it is feasible for an ion with the selectivity properties of NH4(+) to permeate the channel without inducing these changes.

We investigated the effects of changing extracellular K+ concentrations on block of the weak inward-rectifier K+ channel Kir1.1b (ROMK2) by the three intracellular cations Mg2+, Na+, and TEA+. Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K+ in the inside (cytoplasmic) solution and 11 mM K+ in the outside (extracellular) solution, these three cations blocked K+ currents with a range of apparent affinities (Ki (0) = 1.6 mM for Mg2+, 160 mM for Na+, and 1.8 mM for TEA+) but with similar voltage dependence (zδ = 0.58 for Mg2+, 0.71 for Na+, and 0.61 for TEA+) despite having different valences. When external K+ was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg2+ but not for Na+ or TEA+. In contrast, the N152Y mutation increased the affinity for block by TEA+ but not for Na+ or Mg2+. Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg2+ but had a small effect on that by Na+. TEA+ block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K+ could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K+ are explained by shifts in the occupancy of sites within the selectivity filter by K+ ions.

Non-technical summary The inwardly rectifying potassium channel, Kir4.2, is amongst the members of the Kir family of K + channels whose activity is directly regulated by the external potassium concentration. We show here that, rather than increasing expression of the channel protein, this regulation is accomplished by activation of silent channels already present at the cell surface. Understanding the mechanism of this regulation will aid in understanding the physiological processes in which this subset of K + channels is involved, such as regulation of blood pressure and control of neuronal excitability. It may also help to explain some of the consequences of hyperkalaemia, or excessive potassium, in blood plasma. Abstract