Molecular basis of voltage-dependent potassium currents in porcine granulosa cells.

The major objective of this study was to elucidate the molecular bases for K(+) current diversity in porcine granulosa cells (GC). Two delayed rectifier K(+) currents with distinct electrophysiological and pharmacological properties were recorded from porcine GC by using whole-cell patch clamp: 1) a slowly activating, noninactivating current (I(Ks)) antagonized by clofilium, 293B, L-735,821, and L-768,673; and 2) an ultrarapidly activating, slowly inactivating current (I(Kur)) antagonized completely by clofilium and 4-aminopyridine and partially by tetraethylammonium, charybdotoxin, dendrotoxin, and kaliotoxin. The molecular identity of the K(+) channel genes underlying I(Ks) and I(Kur) was examined using reverse transcription-polymerase chain reaction and immunoblotting to detect K(+) channel transcripts and proteins. We found that GC could express multiple voltage-dependent K(+) (Kv) channel subunits, including KCNQ1, KCNE1, Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kvbeta1.3, and Kvbeta2. Coimmunoprecipitation was used to establish the hetero-oligomeric nature of granulosa cell Kv channels. KCNE1 and KCNQ1 were coassociated in GC, and their expression coincided with the expression of I(Ks). Extensive coassociation of the various Kv alpha- and beta-subunits was also documented, suggesting that the diverse electrophysiological and pharmacological properties of I(Kur) currents may reflect variation in the composition and stoichiometry of the channel assemblies, as well as differences in post-translational modification of contributing Kv channel subunits. Our findings provide an essential background for experimental definition of granulosa K(+) channel function(s). It will be critical to define the functional roles of specific GC K(+) channels, because these proteins may represent either novel targets for assisted reproduction or potential sites of drug toxicity.