Sulfonylurea and K(+)-channel opener sensitivity of K(ATP) channels. Functional coupling of Kir6.2 and SUR1 subunits.

The sensitivity of K(ATP) channels to high-affinity block by sulfonylureas and to stimulation by K(+) channel openers and MgADP (PCOs) is conferred by the regulatory sulfonylurea receptor (SUR) subunit, whereas ATP inhibits the channel through interaction with the inward rectifier (Kir6.2) subunit. Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) profoundly antagonized ATP inhibition of K(ATP) channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity. By stabilizing the open state of the channel, PIP(2) drives the channel away from closed state(s) that are preferentially affected by high affinity tolbutamide binding, thereby producing an apparent loss of high affinity tolbutamide inhibition. Mutant K(ATP) channels (Kir6. 2[DeltaN30] or Kir6.2[L164A], coexpressed with SUR1) also displayed an "uncoupled" phenotype with no high affinity tolbutamide block and with intrinsically higher open state stability. Conversely, Kir6. 2[R176A]+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block. In addition to antagonizing high-affinity block by tolbutamide, PIP(2) also altered the stimulatory action of the PCOs, diazoxide and MgADP. With time after PIP(2) application, PCO stimulation first increased, and then subsequently decreased, probably reflecting a common pathway for activation of the channel by stimulatory PCOs and PIP(2). The net effect of increasing open state stability, either by PIP(2) or mutagenesis, is an apparent "uncoupling" of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-L-lysine.     

Endosomal KATP channels as a reservoir after myocardial ischemia: a role for SUR2 subunits

ATP-sensitive K(+) (K(ATP)) channels, composed of inward rectifier K(+) (Kir)6.x and sulfonylurea receptor (SUR)x subunits, are expressed on cellular plasma membranes. We demonstrate an essential role for SUR2 subunits in trafficking K(ATP) channels to an intracellular vesicular compartment. Transfection of Kir6.x/SUR2 subunits into a variety of cell lines (including h9c2 cardiac cells and human coronary artery smooth muscle cells) resulted in trafficking to endosomal/lysosomal compartments, as assessed by immunofluorescence microscopy. By contrast, SUR1/Kir6.x channels efficiently localized to the plasmalemma. The channel turnover rate was similar with SUR1 or SUR2, suggesting that the expression of Kir6/SUR2 proteins in lysosomes is not associated with increased degradation. Surface labeling of hemagglutinin-tagged channels demonstrated that SUR2-containing channels dynamically cycle between endosomal and plasmalemmal compartments. In addition, Kir6.2 and SUR2 subunits were found in both endosomal and sarcolemmal membrane fractions isolated from rat hearts. The balance of these K(ATP) channel subunits shifted to the sarcolemmal membrane fraction after the induction of ischemia. The K(ATP) channel current density was also increased in rat ventricular myocytes isolated from hearts rendered ischemic before cell isolation without corresponding changes in subunit mRNA expression. We conclude that an intracellular pool of SUR2-containing K(ATP) channels exists that is derived by endocytosis from the plasma membrane. In cardiac myocytes, this pool can potentially play a cardioprotective role by serving as a reservoir for modulating surface K(ATP) channel density under stress conditions, such as myocardial ischemia.     

Identification of the PIP2-binding site on Kir6.2 by molecular modelling and functional analysis

ATP-sensitive potassium (K(ATP)) channels couple cell metabolism to electrical activity by regulating K(+) fluxes across the plasma membrane. Channel closure is facilitated by ATP, which binds to the pore-forming subunit (Kir6.2). Conversely, channel opening is potentiated by phosphoinositol bisphosphate (PIP(2)), which binds to Kir6.2 and reduces channel inhibition by ATP. Here, we use homology modelling and ligand docking to identify the PIP(2)-binding site on Kir6.2. The model is consistent with a large amount of functional data and was further tested by mutagenesis. The fatty acyl tails of PIP(2) lie within the membrane and the head group extends downwards to interact with residues in the N terminus (K39, N41, R54), transmembrane domains (K67) and C terminus (R176, R177, E179, R301) of Kir6.2. Our model suggests how PIP(2) increases channel opening and decreases ATP binding and channel inhibition. It is likely to be applicable to the PIP(2)-binding site of other Kir channels, as the residues identified are conserved and influence PIP(2) sensitivity in other Kir channel family members.     

Identification of two types of ATP-sensitive K+ channels in rat ventricular myocytes

The ATP-sensitive K(+) (K(ATP)) channels are known to provide a functional linkage between the electrical activity of the cell membrane and metabolism. Two types of inwardly rectifying K(+) channel subunits (i.e., Kir6.1 and Kir6.2) with which sulfonylurea receptors are associated were reported to constitute the K(ATP) channels. In this study, we provide evidence to show two types of K(ATP) channels with different biophysical properties functionally expressed in isolated rat ventricular myocytes. Using patch-clamp technique, we found that single-channel conductance for the different two types of K(ATP) channels in these cells was 57 and 21 pS. The kinetic properties, including mean open time and bursting kinetics, did not differ between these two types of K(ATP) channels. Diazoxide only activated the small-conductance K(ATP) channel, while pinacidil and dinitrophenol stimulated both channels. Both of these K(ATP) channels were sensitive to block by glibenclamide. Additionally, western blotting, immunochemistry, and RT-PCR revealed two types of Kir6.X channels, i.e., Kir6.1 and Kir6.2, in rat ventricular myocytes. Single-cell Ca(2+) imaging also revealed that similar to dinitrophenol, diazoxide reduced the concentration of intracellular Ca(2+). The present results suggest that these two types of K(ATP) channels may functionally be related to the activity of heart cells.     

Control of pH and PIP2 gating in heteromeric Kir4.1/Kir5.1 channels by H-Bonding at the helix-bundle crossing

Inhibition by intracellular H(+) (pH gating) and activation by phosphoinositides such as PIP(2) (PIP(2)-gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP(2) gating and pH gating are controlled by an intra-subunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP(2) and pH gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP2 gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP(2) affinities. This indicates that Kir channel PIP(2) affinity has little impact on either the PIP(2) or pH gating kinetics.     

A structural determinant for the control of PIP2 sensitivity in G protein-gated inward rectifier K+ channels

Inward rectifier K(+) (Kir) channels are activated by phosphatidylinositol-(4,5)-bisphosphate (PIP(2)), but G protein-gated Kir (K(G)) channels further require either G protein βγ subunits (Gβγ) or intracellular Na(+) for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of K(G) channel subunit Kir3.2 obtained in the presence and the absence of Na(+). The Na(+)-free Kir3.2, but not the Na(+)-plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on the N terminus and Asp-228 on the CD loop, which are known to be critically involved in Gβγ- and Na(+)-dependent activation, lowered PIP(2) sensitivity. The conservation of these residues within the K(G) channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP(2) sensitivity.     

Exaggerated Mg2+ inhibition of Kir2.1 as a consequence of reduced PIP2 sensitivity in Andersen syndrome

Andersen syndrome is an autosomal dominant disorder characterized by cardiac arrhythmias, periodic paralysis and dysmorphic features. Many Andersen syndrome cases have been associated with loss-of-function mutations in the inward rectifier K(+) channel Kir2.1 encoded by KCNJ2. Using engineered concatenated tetrameric channels we determined the mechanism for dominant loss-of-function associated with a trafficking-competent missense mutation, Kir2.1-T74A. This mutation alters a conserved threonine residue in an N-terminal domain analogous to the slide helix identified in the structure of a bacterial inward rectifier. Incorporation of a single mutant subunit in channel tetramers was sufficient to cause a selective impairment of whole-cell outward current, but no difference in the level of inward current compared with wild-type (WT) tetramers. The presence of two mutant subunits resulted in greatly reduced outward and impaired inward currents. Experiments using excised inside-out membrane patches revealed that tetramers with one mutant subunit exhibited increased Mg(2+) inhibition. Additional experiments demonstrated that concatenated tetramers containing one T74A subunit had reduced PIP(2) sensitivity, and that outward current carried by mutant tetramers could be restored by addition of PIP(2) in the absence of Mg(2+). Our results are consistent with the involvement of the Kir2.1 N-terminus in PIP(2) modulation of channel activity and support the existence of an inverse relationship between PIP(2) sensitivity and Mg(2+) inhibition of Kir2.1 channels. Our data also indicate that a single mutant subunit is sufficient to explain dominant-negative behavior of Kir2.1-T74A in Andersen syndrome.     

Direct modulation of Kir channel gating by membrane phosphatidylinositol 4,5-bisphosphate

Multiple ion channels have now been shown to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) at the cytoplasmic face of the membrane. However, direct evidence for a specific interaction between phosphoinositides and ion channels is critically lacking. We reconstituted pure KirBac1.1 and KcsA protein into liposomes of defined composition (3:1 phosphatidylethanolamine:phosphatidylglycerol) and examined channel activity using a 86Rb+ uptake assay. We demonstrate direct modulation by PIP2 of KirBac1.1 but not KcsA activity. In marked contrast to activation of eukaryotic Kir channels by PIP2, KirBac1.1 is inhibited by PIP2 incorporated in the membrane (K(1/2) = 0.3 mol %). The dependence of inhibition on the number of phosphate groups and requirement for a lipid tail matches that for activation of eukaryotic Kir channels, suggesting a fundamentally similar interaction mechanism. The data exclude the possibility of indirect modulation via cytoskeletal or other intermediary elements and establish a direct interaction of the channel with PIP2 in the membrane.     

Alterations in conserved Kir channel-PIP2 interactions underlie channelopathies

Inwardly rectifying K(+) (Kir) channels are important regulators of resting membrane potential and cell excitability. The activity of Kir channels is critically dependent on the integrity of channel interactions with phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here we identify and characterize channel-PIP(2) interactions that are conserved among Kir family members. We find basic residues that interact with PIP(2), two of which have been associated with Andersen's and Bartter's syndromes. We show that several naturally occurring mutants decrease channel-PIP(2) interactions, leading to disease.     

Regulation of cloned ATP-sensitive K channels by adenine nucleotides and sulfonylureas: interactions between SUR1 and positively charged domains on Kir6.2

K(ATP) channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 "PIP2-insensitive" mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the K(ATP) channel. In contrast, the R301C and R314C mutants, which are also "PIP2-insensitive," remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the K(ATP) channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since K(ATP) channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.     

Agonist-induced localization of Gq-coupled receptors and G protein-gated inwardly rectifying K+ (GIRK) channels to caveolae determines receptor specificity of phosphatidylinositol 4,5-bisphosphate signaling

G protein-gated inwardly rectifying K(+) (GIRK) channels are parasympathetic effectors in cardiac myocytes that act as points of integration of signals from diverse pathways. Neurotransmitters and hormones acting on the Gq protein regulate GIRK channels by phosphatidylinositol 4,5-bisphosphate (PIP(2)) depletion. In previous studies, we found that endothelin-1, but not bradykinin, inhibited GIRK channels, even though both of them hydrolyze PIP(2) in cardiac myocytes, showing receptor specificity. The present study assessed whether the spatial organization of the PIP(2) signal into caveolar microdomains underlies the specificity of PIP(2)-mediated signaling. Using biochemical analysis, we examined the localization of GIRK and Gq protein-coupled receptors (GqPCRs) in mouse atrial myocytes. Agonist stimulation induced a transient co-localization of GIRK channels with endothelin receptors in the caveolae, excluding bradykinin receptors. Such redistribution was eliminated by caveolar disruption with methyl-β-cyclodextrin (MβCD). Patch clamp studies showed that the specific response of GIRK channels to GqPCR agonists was abolished by MβCD, indicating the functional significance of the caveolae-dependent spatial organization. To assess whether low PIP(2) mobility is essential for PIP(2)-mediated signaling, we blocked the cytoskeletal restriction of PIP(2) diffusion by latrunculin B. This abolished the GIRK channel regulation by GqPCRs without affecting their targeting to caveolae. These data suggest that without the hindered diffusion of PIP(2) from microdomains, PIP(2) loses its signaling efficacy. Taken together, these data suggest that specific targeting combined with restricted diffusion of PIP(2) allows the PIP(2) signal to be compartmentalized to the targets localized closely to the GqPCRs, enabling cells to discriminate between identical PIP(2) signaling that is triggered by different receptors.     

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Phosphatidylinositol bisphosphate (PIP(2)) is the most abundant phosphoinositide in the plasma membrane of cells and its interaction with many ion channel proteins has proven to be a critical factor enabling ion channel gating. All members of the inwardly rectifying potassium (Kir) channel family depend on PIP(2) for their activity, displaying distinct affinities and stereospecificities of interaction with the phosphoinositide. Here, we explored the stoichiometry of Kir channels with PIP(2). We first showed that PIP(2) regulated the activity of Kir3.4 channels mainly by altering their bursting behavior. Detailed burst analysis indicates that the channels assumed up to four open states and a connectivity of four between open and closed states depending on the available PIP(2) levels. Moreover, by controlling the number of PIP(2)-sensitive subunits in the stoichiometry of a tetrameric Kir2.1 channel, we showed that characteristic channel activity was obtained when at least two wild-type subunits were present. Our studies support a kinetic model for gating of Kir channels by PIP(2), where each of the four open states corresponds to the channel activated by one to four PIP(2) molecules.     

Mechanism of PLC-mediated Kir3 current inhibition

A large number of ion channels maintain their activity through direct interactions with phosphatidylinositol bisphosphate (PIP2). For such channels, hydrolysis of PIP2 causes current inhibition. It has become controversial whether the inhibitory effects on channel activity represent direct effects of PIP2 hydrolysis or of downstream PKC action. We studied Phospholipase C (PLC)-dependent inhibition of G protein-activated inwardly rectifying K+ (Kir3) channels. By monitoring simultaneously channel activity and PIP2 hydrolysis, we determined that both direct PIP2 depletion and PKC actions contribute to Kir3 current inhibition. We show that the PKC-induced effects strongly depend on PIP2 levels in the membrane. At the same time, we show that PKC destabilizes Kir3/PIP2 interactions and enhances the effects of PIP2 depletion on channel activity. These results demonstrate that PIP2 depletion and PKC-mediated effects reinforce each other and suggest that both of these interdependent mechanisms contribute to Kir3 current inhibition. This mechanistic insight may explain how even minor changes in PIP2 levels can have profound effects on Kir3 activity. We also show that stabilization of Kir3/PIP2 interactions by Gbetagamma attenuates both PKC and Gq-mediated current inhibition, suggesting that diverse pathways regulate Kir3 activity through modulation of channel interactions with PIP2.     

Localization of the ATP/phosphatidylinositol 4,5 diphosphate-binding site to a 39-amino acid region of the carboxyl terminus of the ATP-regulated K+ channel Kir1.1

Intracellular ATP and membrane-associated phosphatidylinositol phospholipids, like PIP(2) (PI(4,5)P(2)), regulate the activity of ATP-sensitive K(+) (K(ATP)) and Kir1.1 channels by direct interaction with the pore-forming subunits of these channels. We previously demonstrated direct binding of TNP-ATP (2',3'-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)-ATP) to the COOH-terminal cytosolic domains of the pore-forming subunits of Kir1.1 and Kir6.x channels. In addition, PIP(2) competed for TNP-ATP binding on the COOH termini of Kir1.1 and Kir6.x channels, providing a mechanism that can account for PIP(2) antagonism of ATP inhibition of these channels. To localize the ATP-binding site within the COOH terminus of Kir1.1, we produced and purified maltose-binding protein (MBP) fusion proteins containing truncated and/or mutated Kir1.1 COOH termini and examined the binding of TNP-ATP and competition by PIP(2). A truncated COOH-terminal fusion protein construct, MBP_1.1CDeltaC170, containing the first 39 amino acid residues distal to the second transmembrane domain was sufficient to bind TNP-ATP with high affinity. A construct containing the remaining COOH-terminal segment distal to the first 39 amino acid residues did not bind TNP-ATP. Deletion of 5 or more amino acid residues from the NH(2)-terminal side of the COOH terminus abolished nucleotide binding to the entire COOH terminus or to the first 49 amino acid residues of the COOH terminus. PIP(2) competed TNP-ATP binding to MBP_1.1CDeltaC170 with an EC(50) of 10.9 microm. Mutation of any one of three arginine residues (R188A/E, R203A, and R217A), which are conserved in Kir1.1 and K(ATP) channels and are involved in ATP and/or PIP(2) effects on channel activity, dramatically reduced TNP-ATP binding to MBP_1.1DeltaC170. In contrast, mutation of a fourth conserved residue (R212A) exhibited slightly enhanced TNP-ATP binding and increased affinity for PIP(2) competition of TNP-ATP (EC(50) = 5.7 microm). These studies suggest that the first 39 COOH-terminal amino acid residues form an ATP-PIP(2) binding domain in Kir1.1 and possibly the Kir6.x ATP-sensitive K(+) channels.     

Exaggerated Mg2+ Inhibition of KCNJ2 as a Consequence of Reduced PIP2 Sensitivity in Andersen Syndrome

Andersen syndrome is an autosomal dominant disorder characterized by cardiac arrhythmias, periodic paralysis and dysmorphic features. Many Andersen syndrome cases have been associated with loss-of-function mutations in the inward rectifier K+ channel Kir2.1 encoded by KCNJ2. Using engineered concatenated tetrameric channels we determined the mechanism for dominant loss-of-function associated with a trafficking-competent missense mutation, Kir2.1-T74A. This mutation alters a conserved threonine residue in an N-terminal domain analogous to the slide helix identified in the structure of a bacterial inward rectifier. Incorporation of a single mutant subunit in channel tetramers was sufficient to cause a selective impairment of whole-cell outward current, but no difference in the level of inward current compared with wild-type (WT) tetramers. The presence of two mutant subunits resulted in greatly reduced outward and impaired inward currents. Experiments using excised inside-out membrane patches revealed that tetramers with one mutant subunit exhibited increased Mg2+ inhibition. Additional experiments demonstrated that concatenated tetramers containing one T74A subunit had reduced PIP2 sensitivity, and that outward current carried by mutant tetramers could be restored by addition of PIP2 in the absence of Mg2+. Our results are consistent with the involvement of the Kir2.1 N-terminus in PIP2 modulation of channel activity and support the existence of an inverse relationship between PIP2 sensitivity and Mg2+ inhibition of Kir2.1 channels. Our data also indicate that a single mutant subunit is sufficient to explain dominant-negative behavior of Kir2.1-T74A in Andersen syndrome.     

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Phosphatidylinositol bisphosphate (PIP2) is the most abundant phosphoinositide in the plasma membranes of cells and its interaction with many ion channel proteins has proven to be a critical factor enabling ion channel gating. All members of the inwardly rectifying potassium (Kir) channel family depend on PIP2 for their activity, displaying distinct affinities and stereospecificities of interaction with the phosphoinositide. Here, we explored the stoichiometry of Kir channels with PIP2. We first showed that PIP2 regulated the activity of Kir3.4 channels mainly by altering their bursting behavior. Detailed burst analysis indicates that the channels assumed up to four open states and a connectivity of four between open and closed states depending on the available PIP2 levels. Moreover, by controlling the number of PIP2-sensitive subunits in the stoichiometry of a tetrameric Kir2.1 channel, we showed that characteristic channel activity was obtained when at least two wild-type subunits were present. Our studies support a kinetic model for gating of Kir channels by PIP2, where each of the four open states corresponds to the channel activated by one to four PIP2 molecules.     

Biophysical and molecular mechanisms underlying the modulation of heteromeric Kir4.1-Kir5.1 channels by CO2 and pH

CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4. 1-Kir5.1 were studied in inside-out patches. These Kir4.1-Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P(open)) approximately 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P(open) without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at approximately 1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1-Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P(open) and reduced channel sensitivity to intracellular protons. In the presence of 10 microM PIP2, the Kir4.1-Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1-Kir5.1. In excised patches, interestingly, the Kir4.1-Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.     

Syntaxin-1A actions on sulfonylurea receptor 2A can block acidic pH-induced cardiac K(ATP) channel activation

During cardiac ischemia, ATP stores are depleted, and cardiomyocyte intracellular pH lowers to <7.0. The acidic pH acts on the Kir6.2 subunit of K(ATP) channels to reduce its sensitivity to ATP, causing channel opening. We recently reported that syntaxin-1A (Syn-1A) binds nucleotide binding folds (NBF)-1 and NBF2 of sulfonylurea receptor 2A (SUR2A) to inhibit channel activity (Kang, Y., Leung, Y. M., Manning-Fox, J. E., Xia, F., Xie, H., Sheu, L., Tsushima, R. G., Light, P. E., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 47125-47131). Here, we examined Syn-1A actions on SUR2A to influence the pH regulation of cardiac K(ATP) channels. K(ATP) channel currents from inside-out patches excised from Kir6.2/SUR2A expressing HEK293 cells and freshly isolated cardiac myocytes were increased by reducing intracellular pH from 7.4 to 6.8, which could be blocked by increasing concentrations of Syn-1A added to the cytoplasmic surface. Syn-1A had no effect on C-terminal truncated Kir6.2 (Kir6.2-deltaC26) channels expressed in TSA cells without the SUR subunit. In vitro binding and co-immunoprecipitation studies show that Syn-1A binding to SUR2A or its NBF-1 and NBF-2 domain proteins increased progressively as pH was reduced from 7.4 to 6.0. The enhancement of Syn-1A binding to SUR2A by acidic pH was further regulated by Mg2+ and ATP. Therefore, pH regulates Kir.6.2/SUR2A channels not only by its direct actions on the Kir6.2 subunit but also by modulation of Syn-1A binding to SUR2A. The increased Syn-1A binding to the SUR2A at acidic pH would assert some inhibition of the K(ATP) channels, which may serve as a "brake" to temper the fluctuation of low pH-induced K(ATP) channel opening that could induce fatal reentrant arrhythmias.