Abstract

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_Ksand 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, Kvβ1.3, and Kvβ2. 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 α- and β-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.