Titration of tertiapin-Q inhibition of ROMK1 channels by extracellular protons

Tertiapin-Q (TPN(Q)), a honey bee toxin derivative, inhibits inward-rectifier K(+) channels by binding to their external vestibule. In the present study we found that TPN(Q) inhibition of the channels is profoundly affected by extracellular pH. This pH dependence mainly reflects titration of histidine residue 12 in TPN(Q) by extracellular protons, since it largely vanishes when the histidine residue is replaced with alanine. Not surprisingly, this alanine derivative of TPN(Q) binds to the channel with much lower affinity. Quantitative thermodynamic cycle analysis shows that deprotonation of the histidine residue reduces the TPN(Q)-ROMK1 binding energy by 1.6 kcal/mol. To eliminate pH sensitivity but retain high affinity, we derivatized TPN(Q) by replacing histidine 12 with lysine. This derivative-denoted tertiapin-KQ (TPN(KQ))-not only is practically insensitive to extracellular pH but also binds to the channel with even higher affinity than TPN(Q) at extracellular pH 7.6.     

Synthesis of a stable form of tertiapin: a high-affinity inhibitor for inward-rectifier K+ channels

Tertiapin (TPN), a small protein derived from honey bee venom, inhibits the GIRK1/4 and ROMK1 channels with nanomolar affinities. Methionine residue 13 in TPN interacts with residue F148 in the channel, located just outside of the narrow region of the ROMK1 pore. The methionine residue in TPN can be oxidized by air, which significantly hinders TPN binding to the channels. To overcome the reduction in TPN affinity due to oxidation of M13, we replaced M13 in TPN with fourteen different residues. Out of the fourteen derivatives, only the one in which M13 was replaced by glutamine, TPNQ, binds to the channel with a Ki value very similar to that of native TPN. Since TPNQ is stable and functionally resembles native TPN, it will be a very useful molecular probe for studying the inward-rectifier K+ channels.     

Regulation of Kir channels by intracellular pH and extracellular K(+): mechanisms of coupling

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.     

Molecular coupling in the human ether-a-go-go-related gene-1 (hERG1) K+ channel inactivation pathway

Emerging evidence suggests that K(+) channel inactivation involves coupling between residues in adjacent regions of the channel. Human ether-a-go-go-related gene-1 (hERG1) K(+) channels undergo a fast inactivation gating process that is crucial for maintaining electrical stability in the heart. The molecular mechanisms that drive inactivation in hERG1 channels are unknown. Using alanine scanning mutagenesis, we show that a pore helix residue (Thr-618) that points toward the S5 segment is critical for normal inactivation gating. Amino acid substitutions at position 618 modulate the free energy of inactivation gating, causing enhanced or reduced inactivation. Mutation of an S5 residue that is predicted to be adjacent to Thr-618 (W568L) abolishes inactivation and alters ion selectivity. The introduction of the Thr-618-equivalent residue in Kv1.5 enhances inactivation. Molecular dynamic simulations of the Kv1.2 tetramer reveal van der Waals coupling between hERG1 618- and 568-equivalent residues and a significant increase in interaction energies when threonine is introduced at the 618-equivalent position. We propose that coupling between the S5 segment and pore helix may participate in the inactivation process in hERG1 channels.     

Influence of permeant ions on voltage sensor function in the Kv2.1 potassium channel

We previously demonstrated that the outer vestibule of activated Kv2.1 potassium channels can be in one of two conformations, and that K(+) occupancy of a specific selectivity filter site determines which conformation the outer vestibule is in. These different outer vestibule conformations result in different sensitivities to internal and external TEA, different inactivation rates, and different macroscopic conductances. The [K(+)]-dependent switch in outer vestibule conformation is also associated with a change in rate of channel activation. In this paper, we examined the mechanism by which changes in [K(+)] modulate the rate of channel activation. Elevation of symmetrical [K(+)] or [Rb(+)] from 0 to 3 mM doubled the rate of on-gating charge movement (Q(on)), measured at 0 mV. Cs(+) produced an identical effect, but required 40-fold higher concentrations. All three permeant ions occupied the selectivity filter over the 0.03-3 mM range, so simple occupancy of the selectivity filter was not sufficient to produce the change in Q(on). However, for each of these permeant ions, the speeding of Q(on) occurred with the same concentration dependence as the switch between outer vestibule conformations. Neutralization of an amino acid (K356) in the outer vestibule, which abolishes the modulation of channel pharmacology and ionic currents by the K(+)-dependent reorientation of the outer vestibule, also abolished the K(+)-dependence of Q(on). Together, the data indicate that the K(+)-dependent reorientation in the outer vestibule was responsible for the change in Q(on). Moreover, similar [K(+)]-dependence and effects of mutagenesis indicate that the K(+)-dependent change in rate of Q(on) can account for the modulation of ionic current activation rate. Simple kinetic analysis suggested that K(+) reduced an energy barrier for voltage sensor movement. These results provide strong evidence for a direct functional interaction, which is modulated by permeant ions acting at the selectivity filter, between the outer vestibule of the Kv2.1 potassium channel and the voltage sensor.     

An amino acid triplet in the NH2 terminus of rat ROMK1 determines interaction with SUR2B

ATP-regulated (K(ATP)) channels are formed by an inward rectifier pore-forming subunit (Kir) and a sulfonylurea (glibenclamide)-binding protein, a member of the ATP binding cassette family (sulfonylurea receptor (SUR) or cystic fibrosis transmembrane conductance regulator). The latter is required to confer glibenclamide sensitivity to K(ATP) channels. In the mammalian kidney ROMK1-3 are components of K(ATP) channels that mediate K(+) secretion into urine. ROMK1 and ROMK3 splice variants share the core polypeptide of ROMK2 but also have distinct NH(2)-terminal extensions of 19 and 26 amino acids, respectively. The SUR2B is also expressed in rat kidney tubules and may combine with Kir.1 to form renal K(ATP) channels. Our previous studies showed that co-expression of ROMK2, but not ROMK1 or ROMK3, with rat SUR2B in oocytes generated glibenclamide-sensitive K(+) currents. These data suggest that the NH(2)-terminal extensions in both ROMK1 and ROMK3 block ROMK-SUR2B interaction. Seven amino acids in the NH(2)-terminal extensions of ROMK1 and ROMK3 are identical (amino acids 13-19 in ROMK1 and 20-26 in ROMK3) and may determine ROMK-SUR2B interaction. We constructed a series of hemagglutinin-tagged ROMK1 NH(2)-terminal deletion and substitution mutants and examined glibenclamide-sensitive K(+) currents in oocytes when co-expressed with SUR2B. These studies identified an amino acid triplet "IRA" within the conserved segment in the NH(2) terminus of ROMK1 and ROMK3 that blocks the ability of SUR2B to confer glibenclamide sensitivity to the expressed K(+) currents. The position of this triplet in the ROMK1 NH(2)-terminal extension is also important for the ROMK-SUR2B interactions. In vitro co-translation and immunoprecipitation studies with hemagglutinin-tagged ROMK mutants and SUR2B indicted that direct interaction between these two proteins is required for glibenclamide sensitivity of induced K(+) currents in oocytes. These results suggest that the IRA triplet in the NH(2)-terminal extensions of both ROMK1 and ROMK3 plays a key role in subunit assembly of the renal secretary K(ATP) channel.     

Protein kinase C mediated pH i -regulation of ROMK1 channels via a phosphatidylinositol-4,5-bisphosphate-dependent mechanism

The protein kinase C (PKC) pathway is important for the regulation of K(+) transport. The renal outer medullar K(+) (ROMK1) channels show an exquisite sensitivity to intracellular protons (pH(i)) (effective pK(a) approximately 6.8) and play a key role in K(+) homeostasis during metabolic acidosis. Our molecular dynamic simulation results suggest that PKC-mediated phosphorylation on Thr-193 may disrupt the PIP(2)-channel interaction via a charge-charge interaction between Thr-193 and Arg-188. Therefore, we investigated the role of PKC and pH(i) in regulation of ROMK1 channel activity using a giant patch clamp with Xenopus oocytes expressing wild-type and mutant ROMK1 channels. ROMK1 channels pre-incubated with the PKC activator phorbol-12-myristate-13-acetate exhibited increased sensitivity to pH(i) (effective pK(a) shifted to pH approximately 7.0). In the presence of GF109203X--a PKC selective inhibitor--the effective pK(a) for inhibition of ROMK1 channels by pH(i) decreased (effective pK(a) shifted to pH approximately 6.5). The pH(i) sensitivity of ROMK1 channels mediated by PKC appeared to be dependent of PIP(2) depletion. The giant patch clamp together with site direct mutagenesis revealed that Thr-193 is the phosphorylation site on PKC that regulates the pH(i) sensitivity of ROMK1 channels. Mutation of PKC-induced phosphorylation sites (T193A) decreases the pH(i) sensitivity and increases the interaction of channel-PIP(2). Taken together, these results provide new insights into the molecular mechanisms underlying the pH(i) gating of ROMK1 channel regulation by PKC.     

Localization of the pH gate in Kir1.1 channels

We used cysteine-modifying reagents to localize the pH-sensitive gate in the renal inward-rectifier K(+) channel Kir1.1a (ROMK1). Cytoplasmic-side methanethiosulfonate (MTS) reagents blocked K(+) permeation in native Kir1.1 channels, expressed in Xenopus oocytes. Replacement of three cysteines in the N-terminus, C-terminus, and transmembrane domains eliminated this sensitivity to MTS reagents, as measured with inside-out macropatches. Reintroduction of one cysteine at 175-Kir1.1a in the second transmembrane domain allowed blockade of the open channel by the MTS reagents MTSEA, MTSET, and MTSES and by Ag(+). However, closure of the channel by low pH protected it from modification. Cysteine was also introduced into position G223, which is thought to line the cytoplasmic pore of the channel. MTSET blocked G223C in both the open and closed state. In contrast, MTSEA reduced G223C single-channel conductance from 40 to 23 pS but did not produce complete block. We conclude that cytoplasmic acidification induces a conformational change in the channel protein that prevents access of cysteine-modifying reagents, and presumably also K(+) ions, to the transmembrane pore from the cytoplasm. This is consistent with localization of the Kir1.1 pH gate at the helix bundle crossing near the cytoplasmic end of the transmembrane pore.     

Regulation of ROMK1 channels by protein-tyrosine kinase and -tyrosine phosphatase

We have used the two-electrode voltage clamp technique and the patch clamp technique to investigate the regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and protein-tyrosine kinase (PTK) in oocytes coexpressing ROMK1 and cSrc. Western blot analysis detected the presence of the endogenous PTP-1D isoform in the oocytes. Addition of phenylarsine oxide (PAO), an inhibitor of PTP, reversibly reduced K(+) current by 55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no significant effect on K(+) current in oocytes injected with ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of PTK, increased K(+) current by 120% and completely abolished the effect of PAO in oocytes coexpressing ROMK1 and cSrc. The effects of herbimycin A and PAO were absent in oocytes expressing the ROMK1 mutant R1Y337A in which the tyrosine residue at position 337 was mutated to alanine. However, addition of exogenous cSrc had no significant effect on the activity of ROMK1 channels in inside-out patches. Moreover, the effect of PAO was completely abolished by treatment of oocytes with 20% sucrose and 250 microg/ml concanavalin A, agents that inhibit the endocytosis of ROMK1 channels. Furthermore, the effect of herbimycin A is absent in the oocytes pretreated with either colchicine, an inhibitor of microtubules, or taxol, an agent that freezes microtubules. We conclude that PTP and PTK play an important role in regulating ROMK1 channels. Inhibiting PTP increases the internalization of ROMK1 channels, whereas blocking PTK stimulates the insertion of ROMK1 channels.     

Phosphatidylinositol 4,5-bisphosphate and intracellular pH regulate the ROMK1 potassium channel via separate but interrelated mechanisms

ROMK channels are responsible for K(+) secretion in kidney. The activity of ROMK is regulated by intracellular pH (pH(i)) with acidification causing channel closure (effective pK(a) approximately 6.9). Recently, we and others reported that a direct interaction of the channels with phosphatidyl-4,5-bisphosphate (PIP(2)) is critical for opening of the inwardly rectifying K(+) channels. Here, we investigate the relationship between the mechanisms for regulation of ROMK by PIP(2) and by pH(i). We find that disruption of PIP(2)-ROMK1 interaction not only decreases single-channel open probability (P(o)) but gives rise to a ROMK1 subconductance state. This state has an increased sensitivity to intracellular protons (effective pK(a) shifted to pH approximately 7.8), such that the subconductance channels are relatively quiescent at physiological pH(i). Open probability for the subconductance channels can then be increased by intracellular alkalinization to supra-physiological pH. This increase in P(o) for the subconductance channels by alkalinization is not associated with an increase in PIP(2)-channel interaction. Thus, direct interaction with PIP(2) is critical for ROMK1 to open at full conductance. Disruption of this interaction increases pH(i) sensitivity for the channels via emergence of the subconductance state. The control of open probability of ROMK1 by pH(i) occurs via a mechanism distinct from the regulation by PIP(2).     

Protein kinase C inhibits ROMK1 channel activity via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism

The activity of apical K(+) channels in cortical collecting duct (CCD) is stimulated and inhibited by protein kinase A (PKA) and C (PKC), respectively. Direct interaction between phosphatidylinositol 4,5-bisphosphate (PIP(2)) and the cloned CCD K(+) channel, ROMK1, is critical for channel opening. We have found previously that phosphorylation of ROMK1 by PKA increases affinity of the channel for PIP(2) and mutation of PKA sites reduces the affinity of ROMK1 for PIP(2). In this study we investigate the molecular mechanism for PKC regulation of ROMK and report that mutants of ROMK1 with reduced PIP(2) affinity exhibit an increased sensitivity to inhibition by phorbol myristate acetate (PMA). The effect of PMA can be prevented by pretreatment with calphostin-C. Activation of PKC by carbachol in Xenopus oocytes co-expressing M1 muscarinic receptors also causes inhibition of the channels. Calphostin-C prevents carbachol-induced inhibition, suggesting that activation of PKC is necessary for inhibition of the channels. PMA reduces open probability of the channel in cell-attached patch clamp recordings. After inhibition by PMA in cell-attached recordings, application of PIP(2) to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP(2) content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP(2) content contributes to the inhibition of ROMK1 channels by PKC. This mechanism may underscore the inhibition of K(+) secretion in CCD by hormones that activate PKC.     

Outer pore architecture of a Ca2+-selective TRP channel

The TRP superfamily forms a functionally important class of cation channels related to the product of the Drosophila trp gene. TRP channels display an unusual diversity in activation mechanisms and permeation properties, but the basis of this diversity is unknown, as the structure of these channels has not been studied in detail. To obtain insight in the pore architecture of TRPV6, a Ca(2+)-selective member of the TRPV subfamily, we probed the dimensions of its pore and determined pore-lining segments using cysteine-scanning mutagenesis. Based on the permeability of the channel to organic cations, we estimated a pore diameter of 5.4 A. Mutating Asp(541), a residue involved in high affinity Ca(2+) binding, altered the apparent pore diameter, indicating that this residue lines the narrowest part of the pore. Cysteines introduced in a region preceding Asp(541) displayed a cyclic pattern of reactivity to Ag(+) and cationic methylthio-sulfanate reagents, indicative of a pore helix. The anionic methanethiosulfonate ethylsulfonate showed only limited reactivity in this region, consistent with the presence of a cation-selective filter at the outer part of the pore helix. Based on these data and on homology with the bacterial KcsA channel, we present the first structural model of a TRP channel pore. We conclude that main structural features of the outer pore, namely a selectivity filter preceded by a pore helix, are conserved between K(+) channels and TRPV6. However, the selectivity filter of TRPV6 is wider than that of K(+) channels and lined by amino acid side chains rather than main chain carbonyls.     

KcsA crystal structure as framework for a molecular model of the Na(+) channel pore

The crystal structure of the pore-forming part of the KcsA bacterial K(+)-selective channel suggests a possible motif for related voltage-gated channels. We examined the hypothesis that the spacial orientation of the KcsA M1 and M2 alpha-helices also predicts the backbone location of S5 and S6 helices of the voltage-gated Na(+) channel. That channel's P region structure is expected to be different because selectivity is determined by side-chain interactions rather than by main-chain carbonyls, and its outer vestibule accommodates relatively large toxin molecules, tetrodotoxin (TTX) and saxitoxin (STX), which interact with selectivity ring residues. The Na(+) channel P loop was well-modeled by the alpha-helix-turn-beta-strand motif, which preserves the relationships for toxin interaction with the Na(+) channel found experimentally. This outer vestibule was docked into the extracellular part of the inverted teepee structure formed by the S5 and S6 helices that were spacially located by coordinates of the KcsA M1 and M2 helix main chains [Doyle et al. (1998) Science 280, 69-74], but populated with side chains of the respective S5 and S6 structures. van der Waals contacts were optimized with minimal adjustment of the S5, S6, and P loop structures, forming a densely packed pore structure. Nonregular external S5-P and P-S6 segments were not modeled here, except the P-S6 segment of domain II. The resulting selectivity region structure is consistent with Na(+) channel permeation properties, offering suggestions for the molecular processes involved in selectivity. The ability to construct a Na(+) channel pore model consistent with most of the available biophysical and mutational information suggests that the KcsA structural framework may be conserved in voltage-gated channels.     

Protein kinase C (PKC)-induced phosphorylation of ROMK1 is essential for the surface expression of ROMK1 channels

We carried out in vitro phosphorylation assays to determine whether ROMK1 is a substrate of protein kinase C (PKC) and used the two-electrode voltage clamp method to investigate the role of serine residues 4, 183, and 201, the three putative PKC phosphorylation sites, in the regulation of ROMK1 channel activity. Incubation of the purified His-tagged ROMK1 protein with PKC and radiolabeled ATP resulted in (32)P incorporation into ROMK1 detected by autoradiography. Moreover, the in vitro phosphorylation study of three synthesized peptides corresponding to amino acids 1-16, 174-189, and 196-211 of ROMK1 revealed that serine residues 4 and 201 of ROMK1 were the two main PKC phosphorylation sites. In contrast, (32)P incorporation of peptide 174-189 was absent. In vitro phosphorylation studies with ROMK1 mutants, R1S4/201A, R1S4/183A, and R1S183/201A, demonstrated that the phosphorylation levels of R1S4/201A were significantly lower than those of the other two mutants. Also, the Ba(2+)-sensitive K(+) current in oocytes injected with green fluorescent protein (GFP)-R1S4/201A was only 5% of that in oocytes injected with wild type GFP-ROMK1. In contrast, the K(+) current in oocytes injected with GFP-ROMK1 mutants containing either serine residue 4 or 201 was similar to those injected with wild type ROMK1. Confocal microscope imaging shows that the surface expression of the K(+) channels was significantly diminished in oocytes injected with R1S4/201A and completely absent in oocytes injected with R1S4/183/201A. Furthermore, the biotin labeling technique confirmed that the membrane fraction of ROMK channels was almost absent in HEK293 cells transfected with either R1S4/201A or R1S4/183/201A. However, when serine residues 4 and 201 were mutated to aspartate, the K(+) currents and the surface expression were completely restored. Finally, addition of calphostin C in the incubation medium significantly decreased the K(+) current in comparison with that under control conditions. Biotin labeling technique further indicated that inhibition of PKC decreases the surface ROMK1 expression in human embryonic kidney (HEK) cells transfected with ROMK1. We conclude that ROMK1 is a substrate of PKC and that serine residues 4 and 201 are the two main PKC phosphorylation sites that are essential for the expression of ROMK1 in the cell surface.     

Change of pore helix conformational state upon opening of cyclic nucleotide-gated channels

The structure of the pore region of the alpha subunit of the bovine rod cyclic nucleotide-gated channel was probed using cysteine-scanning mutagenesis and hydrophilic sulfhydryl-reactive methanethiosulfonate (MTS) reagents. A region homologous to the pore helix in the X-ray crystal structure of the KcsA K(+) channel showed a helical pattern of reactivity with externally applied MTS reagents. Surprisingly, three out of four of the reactive residues, all on one face of the pore helix, only reacted with MTS reagents in the closed state. A residue on the opposite face of the helix only reacted with MTS reagents in the open state. These results indicate that the pore helix (or its surroundings) undergoes a change in conformation, perhaps involving a rotation around its long axis, that opens a gate localized to the selectivity filter of the channel.     

Influence of pore residues on permeation properties in the Kv2.1 potassium channel. Evidence for a selective functional interaction of K+ with the outer vestibule

The Kv2.1 potassium channel contains a lysine in the outer vestibule (position 356) that markedly reduces open channel sensitivity to changes in external [K(+)]. To investigate the mechanism underlying this effect, we examined the influence of this outer vestibule lysine on three measures of K(+) and Na(+) permeation. Permeability ratio measurements, measurements of the lowest [K(+)] required for interaction with the selectivity filter, and measurements of macroscopic K(+) and Na(+) conductance, were all consistent with the same conclusion: that the outer vestibule lysine in Kv2.1 interferes with the ability of K(+) to enter or exit the extracellular side of the selectivity filter. In contrast to its influence on K(+) permeation properties, Lys 356 appeared to be without effect on Na(+) permeation. This suggests that Lys 356 limited K(+) flux by interfering with a selective K(+) binding site. Combined with permeation studies, results from additional mutagenesis near the external entrance to the selectivity filter indicated that this site was located external to, and independent from, the selectivity filter. Protonation of a naturally occurring histidine in the same outer vestibule location in the Kv1.5 potassium channel produced similar effects on K(+) permeation properties. Together, these results indicate that a selective, functional K(+) binding site (e.g., local energy minimum) exists in the outer vestibule of voltage-gated K(+) channels. We suggest that this site is the location of K(+) hydration/dehydration postulated to exist based on the structural studies of KcsA. Finally, neutralization of position 356 enhanced outward K(+) current magnitude, but did not influence the ability of internal K(+) to enter the pore. These data indicate that in Kv2.1, exit of K(+) from the selectivity filter, rather than entry of internal K(+) into the channel, limits outward current magnitude. We discuss the implications of these findings in relation to the structural basis of channel conductance in different K(+) channels.     

Carboxy-terminal determinants of conductance in inward-rectifier K channels

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.     

Identification of a titratable lysine residue that determines sensitivity of kidney potassium channels (ROMK) to intracellular pH.

Potassium (K+) homeostasis is controlled by the secretion of K+ ions across the apical membrane of renal collecting duct cells through a low-conductance inwardly rectifying K+ channel. The sensitivity of this channel to intracellular pH is particularly high and assumed to play a key role in K+ homeostasis. Recently, the apical K+ channel has been cloned (ROMK1,2,3 = Kir1.1a, Kir1.1b and Kir1.1c) and the pH dependence of ROMK1 was shown to resemble closely that of the native apical K+ channel. It is reported here that the steep pH dependence of ROMK channels is determined by a single amino acid residue located in the N-terminus close to the first hydrophobic segment M1. Changing lysine (K) at position 80 to methionine (M) removed the sensitivity of ROMK1 channels to intracellular pH. In pH-insensitive IRK1 channels, the reverse mutation (M84K) introduced dependence on intracellular pH similar to that of ROMK1 wild-type. A detailed mutation analysis suggests that a shift in the apparent pKalpha of K80 underlies the pH regulation of ROMK1 channels in the physiological pH range.     

BeKm-1 is a HERG-specific toxin that shares the structure with ChTx but the mechanism of action with ErgTx1

Peptide toxins with disulfide-stabilized structures have been used as molecular calipers to probe the outer vestibule structure of K channels. We want to apply this approach to the human ether-a-go-go-related gene (HERG) channel, whose outer vestibule is unique in structure and function among voltage-gated K channels. Our focus here is BeKm-1, a HERG-specific peptide toxin that can suppress HERG in the low nM concentration range. Although BeKm-1 shares the three-dimensional scaffold with the well-studied charybdotoxin, the two use different mechanisms in suppressing currents through their target K channels. BeKm-1 binds near, but not inside, the HERG pore, and it is possible that BeKm-1-bound HERG channels can conduct currents although with markedly altered voltage-dependence and kinetics of gating. BeKm-1 and ErgTx1 differ in three-dimensional scaffold, but the two share mechanism of action and have overlapping binding sites on the HERG channel. For both, residues in the middle of the S5-P linker (the putative 583-597 helix) and residues at the pore entrance are critical for binding, although specific contact points vary between the two. Toxin foot printing using BeKm-1 and ErgTx1 will likely provide complementary information about the unique outer vestibule structure of the HERG channel.     

Absence of small conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter's) knockout mice

The ROMK (Kir1.1; Kcnj1) gene is believed to encode the apical small conductance K(+) channels (SK) of the thick ascending limb (TAL) and cortical collecting duct (CCD). Loss-of-function mutations in the human ROMK gene cause Bartter's syndrome with renal Na(+) wasting, consistent with the role of this channel in apical K(+) recycling in the TAL that is crucial for NaCl reabsorption. However, the mechanism of renal K(+) wasting and hypokalemia that develop in individuals with ROMK Bartter's syndrome is not apparent given the proposed loss of the collecting duct SK channel. Thus, we generated a colony of ROMK null mice with approximately 25% survival to adulthood that provides a good model for ROMK Bartter's syndrome. The remaining 75% of null mice die in less than 14 days after birth. The surviving ROMK null mice have normal gross renal morphology with no evidence of significant hydronephrosis, whereas non-surviving null mice exhibit marked hydronephrosis. ROMK protein expression was absent in TAL and CCD from null mice but exhibited normal abundance and localization in wild-type littermates. ROMK null mice were polyuric and natriuretic with an elevated hematocrit consistent with mild extracellular volume depletion. SK channel activity in TAL and CCD was assessed by patch clamp analysis in ROMK wild-type ROMK(+/+), heterozygous ROMK(+/-), and null ROMK(-/-) mice. In 313 patches with successful seals from the three ROMK genotypes, SK channel activity in ROMK (+/+ and +/-) exhibited normal single channel kinetics. The expression frequencies are as follows: 67 (TAL) and 58% (CCD) in ROMK(+/+); about half that of the wild-type in ROMK(+/-), being 38 (TAL) and 25% (CCD); absent in both TAL or CCD in ROMK(-/-) between 2 and 5 weeks in 15 mice (61 and 66 patches, respectively). The absence of SK channel activity in ROMK null mice demonstrates that ROMK is essential for functional expression of SK channels in both TAL and CCD. Despite loss of ROMK expression, the normokalemic null mice exhibited significantly increased kaliuresis, indicating alternative mechanisms for K(+) absorption/secretion in the nephron.