Evidence for a Functional Interaction between Integrins and G Protein-activated Inward Rectifier K+ Channels

Heteromultimeric G protein-activated inward rectifier K+ (GIRK) channels, abundant in heart and brain, help to determine the cellular membrane potential as well as the frequency and duration of electrical impulses. The sequence arginine-glycine-aspartate (RGD), located extracellularly between the first membrane-spanning region and the pore, is conserved among all identified GIRK subunits but is not found in the extracellular domain of any other cloned K+ channels. Many integrins, which, like channels, are integral membrane proteins, recognize this RGD sequence on other proteins, usually in the extracellular matrix. We therefore asked whether GIRK activity might be regulated by direct interaction with integrin. Here, we present evidence that mutation of the RGD site to RGE, particularly on the GIRK4 subunit, decreases or abolishes GIRK current. Furthermore, wild-type channels can be co-immunoprecipitated with integrin. The total cellular amount of expressed mutant GIRK channel protein is the same as the wild-type protein; however, the amount of mutant channel protein that localizes to the plasma membrane is decreased relative to wild-type, most likely accounting for the diminished GIRK current detected. GIRK channels appear to bind directly to integrin and to require this interaction for proper GIRK channel membrane localization and function

High affinity binding of pyrethroids to the � subunit of brain sodium channels

SUMMARY Na ϩ channels are the primary molecular targets of the pyrethroid insecticides. Na ϩ channels consisting of only a type IIA ␣ subunit expressed in Chinese hamster ovary cells responded to pyrethroid treatment in a normal manner: a sustained Na ϩ current was induced progressively after each depolarizing pulse in a train of stimuli, and this Na ϩ current decayed slowly on repolarization. These modified Na ϩ channels could be reactivated at much more negative membrane potentials (V 0.5 ϭ Ϫ139 mV) than unmodified Na ϩ channels (V 0.5 ϭ Ϫ28 mV). These results indicate that pyrethroids can modify the functional properties of the Na ϩ channel ␣ subunit expressed alone by blocking their inactivation, shifting their voltage dependence of activation, and slowing their deactivation. To demonstrate directly the specific interaction of pyrethroids with the ␣ subunit of voltage-gated Na ϩ channels, a radioactive photosensitive derivative, [ 3 H]RU58487, was used in binding and photolabeling studies. In the presence of a low concentration of the nonionic detergent Triton X-100, specific pyrethroid binding to Na ϩ channels in rat brain membrane preparations could be measured and reached 75% of total binding under optimal conditions. Binding approached equilibrium within 1 hr at 4°, dissociated with a half-time of ϳ10 min, and had K D values of ϳ58 -300 nM for three representative pyrethroids. Specific pyrethroid binding was enhanced by ϳ40% in the presence of 100 nM ␣-scorpion toxin, but no allosteric enhancement was observed in the presence of toxins acting at other Na ϩ channel receptor sites. Extensive membrane washing increased specific binding to 89%. Photolabeling with [ 3 H]RU58487 under these optimal binding conditions revealed a radiolabeled band with an apparent molecular mass of 240 kDa corresponding to the Na ϩ channel ␣ subunit. Anti-peptide antibodies recognizing sequences within the ␣ subunit were able to specifically immunoprecipitate the covalently modified channel. Together, these results demonstrate that the pyrethroids can modify the properties of cells expressing only the ␣ subunit of Na ϩ channels and can bind specifically to a receptor site on the ␣ subunit