The KCNQ family of potassium channels underlie a repolarizing K ؉ current in the heart and the M-current in neurones.The assembly of KCNQ1 with KCNE1 generates the delayed rectifier current I Ks in the heart. Characteristically these channels are regulated via G q/11 -coupled receptors and the inhibition seen after phospholipase C activation is now thought to occur from membrane phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) depletion. It is not clear how KCNQ1 recognizes PIP 2 and specifically which residues in the channel complex are important. Using biochemical techniques we identify a cluster of basic residues namely, Lys-354, Lys-358, Arg-360, and Lys-362, in the proximal C terminus as being involved in binding anionic phospholipids. The mutation of specific residues in combination, to alanine leads to the loss of binding to phosphoinositides. Functionally, the introduction of these mutations into KCNQ1 leads to shifts in the voltage dependence of channel activation toward depolarized potentials and reductions in current density. Additionally, the biophysical effects of the charge neutralizing mutations, which disrupt phosphoinositide binding, mirror the effects we see on channel function when we deplete cellular PIP 2 levels through activation of a G q/11 -coupled receptor. Conversely, the addition of diC8-PIP 2 to the wild-type channel, but not a PIP 2 binding-deficient mutant, acts to shift the voltage dependence of channel activation toward hyperpolarized potentials and increase current density.In conclusion, we use a combined biochemical and functional approach to identify a cluster of basic residues important for the binding and action of anionic phospholipids on the KCNQ1/KCNE1 complex.

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. ATP-sensitive potassium (K ATP )2 channels couple cell metabolism to membrane potential in many cell types and control insulin release, vascular smooth muscle tone, and excitability in neurons and muscle (1). Inhibition by ATP and activation by nucleotide diphosphates allow the metabolic state of the cell to control membrane potential and cell excitability. Additionally, activation of K ATP channels as a result of metabolic stress such as ischemia and hypoxia has been shown to protect muscle, heart, and brain (2).K ATP channels are composed of an octomeric complex consisting of four pore-forming subunits of the Kir6.x subfamily of inwardly rectifying potassium channels (Kir6.1 or Kir6.2) and four regulatory sulfonylurea receptor subunits (SUR1, SUR2A, or SUR2B), belonging to the ATP-binding cassette superfamily of proteins (1,3,4). The assembly of one pore-forming subunit (Kir6.1 or Kir6.2) with a particular SUR generates currents with distinct nucleotide sensitivities and pharmacological properties (4, 5). For example, the pancreatic ␤-cell K ATP channel is composed of Kir6.2 and SUR1 (6, 7), Kir6.1 and SUR2B are thought to form the vascular smooth muscle K ATP channel (8 -10), Kir6.2 and SUR2B are present in nonvascular smooth muscle and portal vein (11-15), and Kir6.2 and SUR2A comprise the cardiac K ATP channel (16). Characteristically, the vascular smooth muscle K ATP channel has a lower single-channel conductance and has an absolute dependence on nucleotide diphosphates for activity (8). In contrast, in portal vein and colonic smooth muscle cells the single-channel conductance and nucleotide regulation are more compatible with a channel complex containing Kir6.2 (11,15,17).Regulation of proteins by intracellular signals thr...

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