The role of NH2-terminal positive charges in the activity of inward rectifier KATP channels

Approximately half of the NH(2) terminus of inward rectifier (Kir) channels can be deleted without significant change in channel function, but activity is lost when more than approximately 30 conserved residues before the first membrane spanning domain (M1) are removed. Systematic replacement of the positive charges in the NH(2) terminus of Kir6.2 with alanine reveals several residues that affect channel function when neutralized. Certain mutations (R4A, R5A, R16A, R27A, R39A, K47A, R50A, R54A, K67A) change open probability, whereas an overlapping set of mutants (R16A, R27A, K39A, K47A, R50A, R54A, K67A) change ATP sensitivity. Further analysis of the latter set differentiates mutations that alter ATP sensitivity as a consequence of altered open state stability (R16A, K39A, K67A) from those that may affect ATP binding directly (K47A, R50A, R54A). The data help to define the structural determinants of Kir channel function, and suggest possible structural motifs within the NH(2) terminus, as well as the relationship of the NH(2) terminus with the extended cytoplasmic COOH terminus of the channel.     

Stabilization of the activity of ATP-sensitive potassium channels by ion pairs formed between adjacent Kir6.2 subunits

ATP-sensitive potassium (KATP) channels are formed by the coassembly of four Kir6.2 subunits and four sulfonylurea receptor subunits (SUR). The cytoplasmic domains of Kir6.2 mediate channel gating by ATP, which closes the channel, and membrane phosphoinositides, which stabilize the open channel. Little is known, however, about the tertiary or quaternary structures of the domains that are responsible for these interactions. Here, we report that an ion pair between glutamate 229 and arginine 314 in the intracellular COOH terminus of Kir6.2 is critical for maintaining channel activity. Mutation of either residue to alanine induces inactivation, whereas charge reversal at positions 229 and 314 (E229R/R314E) abolishes inactivation and restores the wild-type channel phenotype. The close proximity of these two residues is demonstrated by disulfide bond formation between cysteine residues introduced at the two positions (E229C/R314C); disulfide bond formation abolishes inactivation and stabilizes the current. Using Kir6.2 tandem dimer constructs, we provide evidence that the ion pair likely forms by residues from two adjacent Kir6.2 subunits. We propose that the E229/R314 intersubunit ion pairs may contribute to a structural framework that facilitates the ability of other positively charged residues to interact with membrane phosphoinositides. Glutamate and arginine residues are found at homologous positions in many inward rectifier subunits, including the G-protein-activated inwardly rectifying potassium channel (GIRK), whose cytoplasmic domain structure has recently been solved. In the GIRK structure, the E229- and R314-corresponding residues are oriented in opposite directions in a single subunit such that in the tetramer model, the E229 equivalent residue from one subunit is in close proximity of the R314 equivalent residue from the adjacent subunit. The structure lends support to our findings in Kir6.2, and raises the possibility that a homologous ion pair may be involved in the gating of GIRKs.     

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.     

ATP-sensitive K+-channel subunits on the mitochondria and endoplasmic reticulum of rat cardiomyocytes.

ATP-sensitive K(+) (K(ATP)) channel subunits on the subcellular structures of rat cardiomyocytes were studied with antibodies against Kir6.1 and Kir6.2. According to the results of Western blot analysis, Kir6.1 was strongly expressed in mitochondrial and microsome fractions, and faintly expressed in cell membrane fraction, whereas Kir6.2 was mainly expressed in the microsome fraction and weakly in cell membrane and mitochondrial fractions. Immunohistochemistry showed that Kir6.1 and Kir6.2 were expressed in the endocardium, atrial and ventricular myocardium, and in vascular smooth muscles. Immunoelectron microscopy revealed that Kir6.1 immunoreactivity was mainly localized in the mitochondria, whereas Kir6.2 immunoreactivity was mainly localized in the endoplasmic reticulum and a few in the mitochondria. Both Kir6.1 and Kir6.2 are candidates of mitochondrial K(ATP) channel subunits. The data obtained in this study will be useful for analyzing the composition of K(ATP) channels of cardiomyocytes and help to understanding the cardioprotective role of K(ATP) channels during heart ischemia.     

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.     

Regulation of cardiac inwardly rectifying potassium channels by membrane lipid metabolism

Types and distributions of inwardly rectifying potassium (Kir) channels are one of the major determinants of the electrophysiological properties of cardiac myocytes. Kir2.1 (classical inward rectifier K(+) channel), Kir6.2/SUR2A (ATP-sensitive K(+) channel) and Kir3.1/3.4 (muscarinic K(+) channels) in cardiac myocytes are commonly upregulated by a membrane lipid, phosphatidylinositol 4,5-bisphosphates (PIP(2)). PIP(2) interaction sites appear to be conserved by positively charged amino acid residues and the putative alpha-helix in the C-terminals of Kir channels. PIP(2) level in the plasma membrane is regulated by the agonist stimulation. Kir channels in the cardiac myocytes seem to be actively regulated by means of the change in PIP(2) level rather than by downstream signal transduction pathways.     

Role of Derlin-1 protein in proteostasis regulation of ATP-sensitive potassium channels

ATP-sensitive potassium (K(ATP)) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2 regulate insulin secretion by linking glucose metabolism with membrane potential. The number of K(ATP) channels in the plasma membrane affects the sensitivity of β-cells to glucose. Aberrant surface channel expression leads to insulin secretion disease. Previously, we have shown that K(ATP) channel proteins undergo endoplasmic reticulum (ER)-associated degradation (ERAD) via the ubiquitin-proteasome pathway, and inhibition of proteasome function results in an increase in channel surface expression. Here, we investigated whether Derlin-1, a protein involved in retrotranslocation of misfolded or misassembled proteins across the ER membrane for degradation by cytosolic proteasomes, plays a role in ERAD and, in turn, biogenesis efficiency of K(ATP) channels. We show that both SUR1 and Kir6.2 form a complex with Derlin-1 and an associated AAA-ATPase, p97. Overexpression of Derlin-1 led to a decrease in the biogenesis efficiency and surface expression of K(ATP) channels. Conversely, knockdown of Derlin-1 by RNA interference resulted in increased processing of SUR1 and a corresponding increase in surface expression of K(ATP) channels. Importantly, knockdown of Derlin-1 increased the abundance of disease-causing misfolded SUR1 or Kir6.2 proteins and even partially rescued surface expression in a mutant channel. We conclude that Derlin-1, by being involved in ERAD of SUR1 and Kir6.2, has a role in modulating the biogenesis efficiency and surface expression of K(ATP) channels. The results suggest that physiological or pathological changes in Derlin-1 expression levels may affect glucose-stimulated insulin secretion by altering surface expression of K(ATP) channels.     

Regulation of the ATP-sensitive potassium channel subunit, Kir6.2, by a Ca2+-dependent protein kinase C

The activity of ATP-sensitive potassium (K(ATP)) channels is governed by the concentration of intracellular ATP and ADP and is thus responsive to the metabolic status of the cell. Phosphorylation of K(ATP) channels by protein kinase A (PKA) or protein kinase C (PKC) results in the modulation of channel activity and is particularly important in regulating smooth muscle tone. At the molecular level the smooth muscle channel is composed of a sulfonylurea subunit (SUR2B) and a pore-forming subunit Kir6.1 and/or Kir6.2. Previously, Kir6.1/SUR2B channels have been shown to be inhibited by PKC, and Kir6.2/SUR2B channels have been shown to be activated or have no response to PKC. In this study we have examined the modulation of channel complexes formed of the inward rectifier subunit, Kir6.2, and the sulfonylurea subunit, SUR2B. Using a combination of biochemical and electrophysiological techniques we show that this complex can be inhibited by protein kinase C in a Ca(2+)-dependent manner and that this inhibition is likely to be as a result of internalization. We identify a residue in the distal C terminus of Kir6.2 (Ser-372) whose phosphorylation leads to down-regulation of the channel complex. This inhibitory effect is distinct from activation which is seen with low levels of channel activity.     

Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit

ATP-sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore-forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP-binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C-terminus of one subunit, and with R50 in the N-terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP-dependent channel inhibition. The model also suggests that interactions between the C-terminus of one subunit and the 'slide helix' of the adjacent subunit may be involved in ATP-dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state.     

Molecular Analysis of ATP-sensitive K Channel Gating and Implications for Channel Inhibition by ATP

The β cell K_ATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2ΔC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2ΔC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2ΔC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the K _i is reduced from 175 μM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.     

Phosphoinositides decrease ATP sensitivity of the cardiac ATP-sensitive K(+) channel. A molecular probe for the mechanism of ATP-sensitive inhibition.

Anionic phospholipids modulate the activity of inwardly rectifying potassium channels (Fan, Z., and J.C. Makielski. 1997. J. Biol. Chem. 272:5388-5395). The effect of phosphoinositides on adenosine triphosphate (ATP) inhibition of ATP-sensitive potassium channel (K(ATP)) currents was investigated using the inside-out patch clamp technique in cardiac myocytes and in COS-1 cells in which the cardiac isoform of the sulfonylurea receptor, SUR2, was coexpressed with the inwardly rectifying channel Kir6.2. Phosphoinositides (1 mg/ml) increased the open probability of K(ATP) in low [ATP] (1 microM) within 30 s. Phosphoinositides desensitized ATP inhibition with a longer onset period (>3 min), activating channels inhibited by ATP (1 mM). Phosphoinositides treatment for 10 min shifted the half-inhibitory [ATP] (K(i)) from 35 microM to 16 mM. At the single-channel level, increased [ATP] caused a shorter mean open time and a longer mean closed time. Phosphoinositides prolonged the mean open time, shortened the mean closed time, and weakened the [ATP] dependence of these parameters resulting in a higher open probability at any given [ATP]. The apparent rate constants for ATP binding were estimated to be 0.8 and 0.02 mM(-1) ms(-1) before and after 5-min treatment with phosphoinositides, which corresponds to a K(i) of 35 microM and 5.8 mM, respectively. Phosphoinositides failed to desensitize adenosine inhibition of K(ATP). In the presence of SUR2, phosphoinositides attenuated MgATP antagonism of ATP inhibition. Kir6.2DeltaC35, a truncated Kir6.2 that functions without SUR2, also exhibited phosphoinositide desensitization of ATP inhibition. These data suggest that (a) phosphoinositides strongly compete with ATP at a binding site residing on Kir6.2; (b) electrostatic interaction is a characteristic property of this competition; and (c) in conjunction with SUR2, phosphoinositides render additional, complex effects on ATP inhibition. We propose a model of the ATP binding site involving positively charged residues on the COOH-terminus of Kir6.2, with which phosphoinositides interact to desensitize ATP inhibition.     

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.     

Mutations within the P-loop of Kir6.2 modulate the intraburst kinetics of the ATP-sensitive potassium channel

The ATP-sensitive potassium (K(ATP)) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 K(ATP) channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (tau(o)) and the short closed time (tauC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer tau(o), shorter tauC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the K(ATP) channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings.     

Mechanism of ATP-sensitive K Channel Inhibition by Sulfhydryl Modification

ATP-sensitive potassium (K_ATP) channels are reversibly inhibited by intracellular ATP. Agents that interact with sulfhydryl moieties produce an irreversible inhibition of K_ATP channel activity when applied to the intracellular membrane surface. ATP appears to protect against this effect, suggesting that the cysteine residue with which thiol reagents interact may either lie within the ATP-binding site or be inaccessible when the channel is closed. We have examined the interaction of the membrane-impermeant thiol-reactive agent p-chloromercuriphenylsulphonate (pCMPS) with the cloned β cell K_ATP channel. This channel comprises the pore-forming Kir6.2 and regulatory SUR1 subunits. We show that the cysteine residue involved in channel inhibition by pCMPS resides on the Kir6.2 subunit and is located at position 42, which lies within the NH₂ terminus of the protein. Although ATP protects against the effects of pCMPS, the ATP sensitivity of the K_ATP channel was unchanged by mutation of C42 to either valine (V) or alanine (A), suggesting that ATP does not interact directly with this residue. These results are consistent with the idea that C42 is inaccessible to the intracellular solution, and thereby protected from interaction with pCMPS when the channel is closed by ATP. We also observed that the C42A mutation does not affect the ability of SUR1 to endow Kir6.2 with diazoxide sensitivity, and reduces, but does not prevent, the effects of MgADP and tolbutamide, which are mediated via SUR1. The Kir6.2-C42A (or V) mutant channel may provide a suitable background for cysteine-scanning mutagenesis studies.     

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.     

Concerted gating mechanism underlying KATP channel inhibition by ATP

KATP channels assemble from four regulatory SUR1 and four pore-forming Kir6.2 subunits. At the single-channel current level, ATP-dependent gating transitions between the active burst and the inactive interburst conformations underlie inhibition of the KATP channel by intracellular ATP. Previously, we identified a slow gating mutation, T171A in the Kir6.2 subunit, which dramatically reduces rates of burst to interburst transitions in Kir6.2DeltaC26 channels without SUR1 in the absence of ATP. Here, we constructed all possible mutations at position 171 in Kir6.2DeltaC26 channels without SUR1. Only four substitutions, 171A, 171F, 171H, and 171S, gave rise to functional channels, each increasing Ki,ATP for ATP inhibition by >55-fold and slowing gating to the interburst by >35-fold. Moreover, we investigated the role of individual Kir6.2 subunits in the gating by comparing burst to interburst transition rates of channels constructed from different combinations of slow 171A and fast T171 "wild-type" subunits. The relationship between gating transition rate and number of slow subunits is exponential, which excludes independent gating models where any one subunit is sufficient for inhibition gating. Rather, our results support mechanisms where four ATP sites independently can control a single gate formed by the concerted action of all four Kir6.2 subunit inner helices of the KATP channel.     

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.     

The carboxyl termini of K(ATP) channels bind nucleotides

ATP-sensitive potassium (K(ATP)) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of K(ATP) channel activity may involve direct binding of this nucleotide to the pore-forming inward rectifier (Kir) subunit despite the lack of known nucleotide-binding motifs. To examine this possibility, we assessed the binding of the fluorescent ATP analogue, 2',3'-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)adenosine 5'-triphosphate (TNP-ATP) to maltose-binding fusion proteins of the NH(2)- and COOH-terminal cytosolic regions of the three known K(ATP) channels (Kir1.1, Kir6.1, and Kir6.2) as well as to the COOH-terminal region of an ATP-insensitive inward rectifier K(+) channel (Kir2.1). We show direct binding of TNP-ATP to the COOH termini of all three known K(ATP) channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of K(ATP) channels because this nucleotide did not bind to the NH(2) termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to K(ATP) COOH termini of Kir1.1, Kir6.1, and Kir6.2 were similar. Binding was abolished by denaturing with 4 m urea or SDS and enhanced by reduction in pH. TNP-ATP to protein stoichiometries were similar for all K(ATP) COOH-terminal proteins with 1 mol of TNP-ATP binding/mole of protein. Competition of TNP-ATP binding to the Kir1.1 COOH terminus by MgATP was complex with both Mg(2+) and MgATP effects. Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of K(ATP) tetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer.     

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.     

Critical protein domains and amino acid residues for gating the KIR6.2 channel by intracellular ATP

K(ATP) channels couple intermediary metabolism to cellular excitability. Such a property relies on the inherent ATP-sensing mechanism known to be located in the Kir6 subunit. However, the molecular basis for the ATP sensitivity remains unclear. Here we showed evidence for protein domains and amino acid residues essential for the channel gating by intracellular ATP. Chimerical channels were constructed using protein domains of Kir6.2 and Kir1.1, expressed in HEK293 cells, and studied in inside-out patches. The N and C termini, although important, were inadequate for channel gating by intracellular ATP. Full ATP sensitivity also required M1 and M2 helices. Cytosolic portions of the M1 and M2 sequences were crucial, in which six amino acid residues were identified, i.e., Thr76, Met77, Ala161, Iso162, Leu164, and Cys166. Site-specific mutation of any of them reduced the ATP sensitivity. Construction of these residues together with the N/C termini produced ATP sensitivity identical to the wild-type channels. The requirement for specific membrane helices suggests that the Kir6.2 gating by ATP is not shared by even two closest relatives in the K(+) channel family, although the general gating mechanisms involving membrane helices appear to be conserved in all K(+) channels.