Ion channels

Abstract:

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple electrical activity to cellular metabolism through their inhibition by intracellular ATP. ATP inhibition of KATP channels varies among tissues and is affected by the metabolic and regulatory state of individual cells, suggesting involvement of endogenous factors. It is reported here that phosphatidylinositol-4, 5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) controlled ATP inhibition of cloned KATP channels (Kir6.2 and SUR1). These phospholipids acted on the Kir6.2 subunit and shifted ATP sensitivity by several orders of magnitude. Receptor-mediated activation of phospholipase C resulted in inhibition of KATP-mediated currents. These results represent a mechanism for control of excitability through phospholipids.

Abstract:

ATP-sensitive potassium (KATP) channels are reversibly inhibited by intracellular ATP. Agents that interact with sulfhydryl moieties produce an irreversible inhibition of KATP 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 beta cell KATP 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 KATP 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.

Abstract:

The beta cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2DeltaC26), 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 Kir6.2DeltaC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2DeltaC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 microM 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.

Abstract:

Sulfonylureas stimulate insulin secretion from pancreatic beta-cells by closing ATP-sensitive K+ (K(ATP)). The beta-cell and cardiac muscle K(ATP) channels have recently been cloned and shown to possess a common pore-forming subunit (Kir6.2) but different sulfonylurea receptor subunits (SUR1 and SUR2A, respectively). We examined the mechanism underlying the tissue specificity of the sulfonylureas tolbutamide and glibenclamide, and the benzamido-derivative meglitinide, using cloned beta-cell (Kir6.2/SUR1) and cardiac (Kir6.2/SUR2A) K(ATP) channels expressed in Xenopus oocytes. Tolbutamide inhibited Kir6.2/SUR1 (Ki approximately 5 micromol/l), but not Kir6.2/SUR2A, currents with high affinity. Meglitinide produced high-affinity inhibition of both Kir6.2/SUR1 and Kir6.2/SUR2A currents (Kis approximately 0.3 micromol/l and approximately 0.5 micromol/l, respectively). Glibenclamide also blocked Kir6.2/SUR1 and Kir6.2/SUR2A currents with high affinity (Kis approximately 4 nmol/l and approximately 27 nmol/l, respectively); however, only for cardiac-type K(ATP) channels was this block reversible. Physiological concentrations of MgADP (100 micromol/l) enhanced glibenclamide inhibition of Kir6.2/SUR1 currents but reduced that of Kir6.2/SUR2A currents. The results suggest that SUR1 may possess separate high-affinity binding sites for sulfonylurea and benzamido groups. SUR2A, however, either does not possess a binding site for the sulfonylurea group or is unable to translate the binding at this site into channel inhibition. Although MgADP reduces the inhibitory effect of glibenclamide on cardiac-type K(ATP) channels, drugs that bind to the common benzamido site have the potential to cause side effects on the heart.