ABSTRACT: The effect of external potassium (K) and cesium (Cs) on the inwardly rectifying K channel ROMK2 (K(ir)1.1b) was studied in Xenopus oocytes. Elevating external K from 1 to 10 mM increased whole-cell outward conductance by a factor of 3.4 +/- 0.4 in 15 min and by a factor of 5.7 +/- 0.9 in 30 min (n = 22). Replacing external Na by Cs blocked inward conductance but increased whole-cell conductance by a factor of 4.5 +/- 0.5 over a period of 40 min (n = 15). In addition to this slow increase in conductance, there was also a small, rapid increase in conductance that occurred as soon as ROMK was exposed to external cesium or 10 mM K. This rapid increase could be explained by the observed increase in ROMK single-channel conductance from 6.4 +/- 0.8 pS to 11.1 +/- 0.8 pS (10 mM K, n = 8) or 11.7 +/- 1.2 pS (Cs, n = 8). There was no effect of either 10 mM K or cesium on the high open probability (P(o) = 0.97 +/- 0.01; n = 12) of ROMK outward currents. In patch-clamp recordings, the number of active channels increased when the K concentration at the outside surface was raised from 1 to 50 mM K. In cell-attached patches, exposure to 50 mM external K produced one or more additional channels in 9/16 patches. No change in channel number was observed in patches continuously exposed to 50 mM external K. Hence, the slow increase in whole-cell conductance is interpreted as activation of pre-existing ROMK channels that had been inactivated by low external K. This type of time-dependent channel activation was not seen with IRK1 (K(ir)2.1) or in ROMK2 mutants in which any one of 6 residues, F129, Q133, E132, V121, L117, or K61, were replaced by their respective IRK1 homologs. These results are consistent with a model in which ROMK can exist in either an activated mode or an inactivated mode. Within the activated mode, individual channels undergo rapid transitions between open and closed states. High (10 mM) external K or Cs stabilizes the activated mode, and low external K stabilizes the inactivated mode. Mutation of a pH-sensing site (ROMK2-K61) prevents transitions from activated to inactivated modes. This is consistent with a direct effect of external K or Cs on the gating of ROMK by internal pH.
Project description:The structural domains contributing to ion permeation and selectivity in K channels were examined in inward-rectifier K(+) channels ROMK2 (Kir1.1b), IRK1 (Kir2.1), and their chimeras using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode with different permeant cations in the pipette. For inward K(+) conduction, replacing the extracellular loop of ROMK2 with that of IRK1 increased single-channel conductance by 25 pS (from 39 to 63 pS), whereas replacing the COOH terminus of ROMK2 with that of IRK1 decreased conductance by 16 pS (from 39 to 22 pS). These effects were additive and independent of the origin of the NH(2) terminus or transmembrane domains, suggesting that the two domains form two resistors in series. The larger conductance of the extracellular loop of IRK1 was attributable to a single amino acid difference (Thr versus Val) at the 3P position, three residues in front of the GYG motif. Permeability sequences for the conducted ions were similar for the two channels: Tl(+) > K(+) > Rb(+) > NH(4)(+). The ion selectivity sequence for ROMK2 based on conductance ratios was NH(4)(+) (1.6) > K(+) (1) > Tl(+) (0.5) > Rb(+) (0.4). For IRK1, the sequence was K(+) (1) > Tl(+) (0.8) > NH(4)(+) (0.6) >> Rb(+) (0.1). The difference in the NH(4)(+)/ K(+) conductance (1.6) and permeability (0.09) ratios can be explained if NH(4)(+) binds with lower affinity than K(+) to sites within the pore. The relatively low conductances of NH(4)(+) and Rb(+) through IRK1 were again attributable to the 3P position within the P region. Site-directed mutagenesis showed that the IRK1 selectivity pattern required either Thr or Ser at this position. In contrast, the COOH-terminal domain conferred the relatively high Tl(+) conductance in IRK1. We propose that the P-region and the COOH terminus contribute independently to the conductance and selectivity properties of the pore.
Project description:The gating characteristics of two ion channels in the inward-rectifier K+ channel superfamily were compared at the single-channel level. The strong inward rectifier IRK1 (Kir 2.1) opened and closed with kinetics that were slow relative to those of the weakly rectifying ROMK2 (Kir 1.1b). At a membrane potential of -60 mV, both IRK and ROMK had single-exponential open-time distributions, with mean open times of 279 +/- 58 ms (n = 4) for IRK1 and 23 +/- 1 ms (n = 7) for ROMK. At -60 mV (and no EDTA) ROMK2 had two closed times: 1.3 +/- 0.1 and 36 +/- 3 ms (n = 7). Under the same conditions, IRK1 exhibited four discrete closed states with mean closed times of 0.8 +/- 0.1 ms, 14 +/- 0.6 ms, 99 +/- 19 ms, and 2744 +/- 640 ms (n = 4). Both the open and the three shortest closed-time constants of IRK1 decreased monotonically with membrane hyperpolarization. IRK1 open probability (Po) decreased sharply with hyperpolarization due to an increase in the frequency of long closed events that were attributable to divalent-cation blockade. Chelation of divalent cations with EDTA eliminated the slowest closed-time distribution of IRK1 and blunted the hyperpolarization-dependent fall in open probability. In contrast, ROMK2 had shorter open and closed times and only two closed states, and its Po was less affected by hyperpolarization. Chimeric channels were constructed to address the question of which parts of the molecules were responsible for the differences in kinetics. The property of multiple closed states was conferred by the second membrane-spanning domain (M2) of IRK. The long-lived open and closed states, including the higher sensitivity to extracellular divalent cations, correlated with the extracellular loop of IRK, including the "P-region." Channel kinetics were essentially unaffected by the N- and C-termini. The data of the present study are consistent with the idea that the locus of gating is near the outer mouth of the pore.
Project description: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.
Project description:Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at V(m) = -100 mV from 34 to 21 pS. The slope conductance measured between -60 and -140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba(2+), suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl(+):K(+), accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K(+) transport.
Project description:ROMK channels are regulated by internal pH (pH(i)) and extracellular K(+) (K(+)(o)). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H(+), increasing the apparent pKa for inhibition by approximately 0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K(+)(o), consistent with coupling between the responses to pH(i) and K(+)(o). The ion selectivity sequence of the activation of the channel by cations was K(+) congruent with Rb(+) > NH(4)(+) >> Na(+), similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K(+) from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K(+)(o). We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K(+)(o) between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K(+) to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba(2+) block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H(+). We conclude that decreasing pH(I) increases the sensitivity of ROMK2 channels to K(+)(o) by altering the properties of the selectivity filter.
Project description:An increased flux of potassium ions into the mitochondrial matrix through the ATP-sensitive potassium channel (mitoK<sub>ATP</sub>) has been shown to provide protection against ischemia-reperfusion injury. Recently, it was proposed that the mitochondrial-targeted isoform of the renal outer medullary potassium channel (ROMK) protein creates a pore-forming subunit of mitoK<sub>ATP</sub> in heart mitochondria. Our research focuses on the properties of mitoK<sub>ATP</sub> from heart-derived H9c2 cells. For the first time, we detected single-channel activity and describe the pharmacology of mitoK<sub>ATP</sub> in the H9c2 heart-derived cells. The patch-clamping of mitoplasts from wild type (WT) and cells overexpressing ROMK2 revealed the existence of a potassium channel that exhibits the same basic properties previously attributed to mitoK<sub>ATP</sub>. ROMK2 overexpression resulted in a significant increase of mitoK<sub>ATP</sub> activity. The conductance of both channels in symmetric 150/150 mM KCl was around 97 ± 2 pS in WT cells and 94 ± 3 pS in cells overexpressing ROMK2. The channels were inhibited by 5-hydroxydecanoic acid (a mitoK<sub>ATP</sub> inhibitor) and by Tertiapin Q (an inhibitor of both the ROMK-type channels and mitoK<sub>ATP</sub>). Additionally, mitoK<sub>ATP</sub> from cells overexpressing ROMK2 were inhibited by ATP/Mg<sup>2+</sup> and activated by diazoxide. We used an assay based on proteinase K to examine the topology of the channel in the inner mitochondrial membrane and found that both termini of the protein localized to the mitochondrial matrix. We conclude that the observed activity of the channel formed by the ROMK protein corresponds to the electrophysiological and pharmacological properties of mitoK<sub>ATP</sub>.
Project description:Tamm-Horsfall glycoprotein (THGP) or Uromodulin is a membrane protein exclusively expressed along the thick ascending limb (TAL) and early distal convoluted tubule (DCT) of the nephron. Mutations in the THGP encoding gene result in Familial Juvenile Hyperuricemic Nephropathy (FJHN), Medullary Cystic Kidney Disease type 2 (MCKD-2), and Glomerulocystic Kidney Disease (GCKD). The physicochemical and biological properties of THGP have been studied extensively, but its physiological function in the TAL remains obscure. We performed yeast two-hybrid screening employing a human kidney cDNA library and identified THGP as a potential interaction partner of the renal outer medullary potassium channel (ROMK2), a key player in the process of salt reabsorption along the TAL. Functional analysis by electrophysiological techniques in Xenopus oocytes showed a strong increase in ROMK current amplitudes when co-expressed with THGP. The effect of THGP was specific for ROMK2 and did not influence current amplitudes upon co-expression with Kir2.x, inward rectifier potassium channels related to ROMK. Single channel conductance and open probability of ROMK2 were not altered by co-expression of THGP, which instead increased surface expression of ROMK2 as determined by patch clamp analysis and luminometric surface quantification, respectively. Despite preserved interaction with ROMK2, disease-causing THGP mutants failed to increase its current amplitude and surface expression. THGP(-/-) mice exhibited increased ROMK accumulation in intracellular vesicular compartments when compared with WT animals. Therefore, THGP modulation of ROMK function confers a new role of THGP on renal ion transport and may contribute to salt wasting observed in FJHN/MCKD-2/GCKD patients.
Project description:Activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) has been implicated in the mechanism of cardiac ischemic preconditioning, yet its molecular composition is unknown.To use an unbiased proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K(+) channel underlying mitoK(ATP).Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and ROMK mRNA was confirmed to be present in neonatal rat ventricular myocytes and adult hearts. ROMK2, a short form of the channel, is shown to contain an N-terminal mitochondrial targeting signal, and a full-length epitope-tagged ROMK2 colocalizes with mitochondrial ATP synthase ?. The high-affinity ROMK toxin, tertiapin Q, inhibits mitoK(ATP) activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, short hairpin RNA-mediated knockdown of ROMK inhibits the ATP-sensitive, diazoxide-activated component of mitochondrial thallium uptake. Finally, the heart-derived cell line, H9C2, is protected from cell death stimuli by stable ROMK2 overexpression, whereas knockdown of the native ROMK exacerbates cell death.The findings support ROMK as the pore-forming subunit of the cytoprotective mitoK(ATP) channel.
Project description:Cells in taste buds are closely packed, with little extracellular space. Tight junctions and other barriers further limit permeability and may result in buildup of extracellular K(+) following action potentials. In many tissues, inwardly rectifying K channels such as the renal outer medullary K (ROMK) channel (also called Kir1.1 and derived from the Kcnj1 gene) help to redistribute K(+). Using reverse-transcription polymerase chain reaction (RT-PCR), we defined ROMK splice variants in mouse kidney and report here the expression of a single one of these, ROMK2, in a subset of mouse taste cells. With quantitative (q)RT-PCR, we show the abundance of ROMK mRNA in taste buds is vallate > foliate > > palate > > fungiform. ROMK protein follows the same pattern of prevalence as mRNA, and is essentially undetectable by immunohistochemistry in fungiform taste buds. ROMK protein is localized to the apical tips of a subset of taste cells. Using tissues from PLCbeta2-GFP and GAD1-GFP transgenic mice, we show that ROMK is not found in PLCbeta2-expressing type II/receptor cells or in GAD1-expressing type III/presynaptic cells. Instead, ROMK is found, by single-cell RT-PCR and immunofluorescence, in most cells that are positive for the taste glial cell marker, Ectonucleotidase2. ROMK is precisely localized to the apical tips of these cells, at and above apical tight junctions. We propose that in taste buds, ROMK in type I/glial-like cells may serve a homeostatic function, excreting excess K(+) through the apical pore, and allowing excitable taste cells to maintain a hyperpolarized resting membrane potential.
Project description:ROMK (Kir1.1) potassium channels are closed by internal acidification with a pKa of 6.7 +/- 0.01 in 100 mM external K and a pKa of 7.0 +/- 0.01 in 1 mM external K. Internal acidification in 1 mM K (but not 100 mM K) not only closed the pH gate but also inactivated Kir1.1, such that realkalization did not restore channel activity until high K was returned to the bath. We identified a new putative intersubunit salt bridge (R128-E132-Kir1.1b) in the P-loop of the channel near the selectivity filter that affected the K sensitivity of the inactivation process. Mutation of either R128-Kir1.1b or E132-Kir1.1b caused inactivation in both 1 mM and 100 mM external K during oocyte acidification. However, 300 mM external K (but not 200 mM Na + 100 mM K) protected both E132Q and R128Y from inactivation. External application of a modified honey-bee toxin, tertiapin Q (TPNQ), also protected Kir1.1 from inactivation in 1 mM K and protected E132Q and R128Y from inactivation in 100 mM K, which suggests that TPNQ binding to the outer mouth of the channel stabilizes the active state. Pretreatment of Kir1.1 with external Ba prevented Kir1.1 inactivation, similar to pretreatment with TPNQ. In addition, mutations that disrupted transmembrane helix H-bonding (K61M-Kir1.1b) or stabilized a selectivity filter to helix-pore linkage (V121T-Kir1.1b) also protected both E132Q and R128Y from inactivation in 1 mM K and 100 mM K. Our results are consistent with Kir inactivation arising from conformational changes near the selectivity filter, analogous to C-type inactivation.