ATP-sensitive (KATP) channels are present in the surface and internal membranes of cardiac, skeletal and smooth muscle cell, and provide a unique feedback between muscle cell metabolism and electrical activity. In so doing, they can play an important role in the control of contractility, particularly when cellular energetics are compromised, protecting the tissue against calcium overload and fiber damage, but the cost of this protection may be enhanced arrhythmic activity. Generated as complexes of Kir6.1 or Kir6.2 pore-forming subunits with regulatory sulfonylurea receptor subunits, SUR1 or SUR2, the differential assembly of KATP channels in different tissues gives rise to tissue-specific physiological and pharmacological regulation, and hence to the tissue-specific pharmacological control of contractility. The last ten years have provided insights to the regulation and role of muscle KATP channels, in large part driven by studies of mice in which the protein determinants of channel activity have been deleted or modified. As yet, few human diseases have been correlated with altered muscle KATP activity, but genetically modified animals give important insights to likely pathological roles of aberrant channel activity in different muscle types.

K(ATP) channels can be formed from Kir6.2 subunits with or without SUR1. The open-state stability of K(ATP) channels can be increased or reduced by mutations throughout the Kir6.2 subunit, and is increased by application of PIP(2) to the cytoplasmic membrane. Increase of open-state stability is manifested as an increase in the channel open probability in the absence of ATP (Po(zero)) and a correlated decrease in sensitivity to inhibition by ATP. Single channel lifetime analyses were performed on wild-type and I154C mutant channels expressed with, and without, SUR1. Channel kinetics include a single, invariant, open duration; an invariant, brief, closed duration; and longer closed events consisting of a "mixture of exponentials," which are prolonged in ATP and shortened after PIP(2) treatment. The steady-state and kinetic data cannot be accounted for by assuming that ATP binds to the channel and causes a gate to close. Rather, we show that they can be explained by models that assume the following regarding the gating behavior: 1) the channel undergoes ATP-insensitive transitions from the open state to a short closed state (C(f)) and to a longer-lived closed state (C(0)); 2) the C(0) state is destabilized in the presence of SUR1; and 3) ATP can access this C(0) state, stabilizing it and thereby inhibiting macroscopic currents. The effect of PIP(2) and mutations that stabilize the open state is then to shift the equilibrium of the "critical transition" from the open state to the ATP-accessible C(0) state toward the O state, reducing accessibility of the C(0) state, and hence reducing ATP sensitivity.

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The glycine-tyrosine-glycine (GYG) sequence in the p-loop of K+ channel subunits lines a narrow pore through which K+ ions pass in single file intercalated by water molecules. Mutation of the motif can give rise to non-selective channels, but it is clear that other structural features are also required for selectivity because, for instance, a recently identified class of cyclic nucleotide-gated pacemaker channels has the GYG motif but are poorly K+ selective. We show that mutation of charged glutamate and arginine residues behind the selectivity filter in the Kir3.1/Kir3.4 K+ channel reduces or abolishes K+ selectivity, comparable with previously reported effects in the Kir2.1 K+ channel. It has been suggested that a salt bridge exists between the glutamate-arginine residue pair. Molecular modeling indicates that the salt bridge does exist, and that it acts as a "bowstring" to maintain the rigid bow-like structure of the selectivity filter and restrict selectivity to K+. The modeling shows that relaxation of the bowstring by mutation of the residue pair leads to enhanced flexibility of the p-loop, allowing permeation of other cations, including polyamines. In experiments, mutation of the residue pair can also abolish polyamine-induced inward rectification. The latter effect occurs because polyamines now permeate rather than block the channel, to the remarkable extent that large polyamine currents can be measured.

Multiple ion channels have now been shown to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 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 86 Rb ؉ uptake assay. We demonstrate direct modulation by PIP 2 of KirBac1.1 but not KcsA activity. In marked contrast to activation of eukaryotic Kir channels by PIP 2 , KirBac1.1 is inhibited by PIP 2 incorporated in the membrane (K1 ⁄ 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 PIP 2 in the membrane.Phosphoinosotides constitute a major group of signaling molecules in eukaryotic membranes (1, 2) and modulate an ever growing list of ion channels, whether by application of exogenous phospholipids to the cytoplasmic membrane surface or by manipulation of endogenous phospholipids (3-12). However, the nature of the phosphoinositide-channel interaction remains elusive. For one extensively studied group, the inward rectifier K (Kir) channels, there is an emerging consensus that a direct interaction occurs between cytoplasmic domains of the channel and inositol headgroups, based on electrophysiological analysis (5, 13-16) and biochemical analysis of isolated channel domains (5,17,18). Direct interaction of functional channels with phospholipids in the membrane has been difficult to demonstrate unequivocally, and without this, quantification of the dose-response relationships for channel modulation by phospholipids is obviated, and further mechanistic understanding is limited (19, 20).The recent cloning and crystallization (21), as well as functional analysis of KirBac1.1 channels reconstituted in lipid membranes (22), provides the opportunity to examine channel activity using a highly purified protein preparation in membranes of defined composition and permits direct test of the nature of the channel-phosphoinositide interaction. EXPERIMENTAL PROCEDURESMethods are essentially as described previously (22). KcsA and KirBac1.1 in pQE60 vector were expressed in BL21* (DE3) cells after induction with isopropyl ␤-D-thiogalactopyranoside. Bacteria were lysed by sonication, incubated 2-4 h with 30 mM decylmaltoside (Anatrace), then centrifuged at 30,000 ϫ g for 30 min, and the supernatant was applied to a cobalt affinity column. The column was washed with 20 -30 volumes of wash buffer (50 mM Tris-HCl, pH 8.0, 150 mM KCl, 10 mM imidazole, and 5 mM decylmaltoside) and eluted with 1-2 ml of wash buffer containing 500 mM imidazole. Protein was concentrated u...