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Mason DE, Mitchell KE, Li Y, Finley MR, Freeman LC: Molecular basis of voltage-dependent potassium currents in porcine granulosa cells. Mol Pharmacol. 2002 Jan;61(1):201-13.
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. |
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