Molecular Basis of Voltage-Dependent Potassium Currents in Porcine Granulosa Cells DIANE E. MASON, KATHY E. MITCHELL, YAN LI, MELISSA R. FINLEY, and LISA C. FREEMAN Departments of Anatomy and Physiology (K.E.M., Y.L., M.R.F., L.C.F.) and Clinical Sciences (D.E.M.), College of Veterinary Medicine, Kansas State University, Manhattan, Kansas Received March 9, 2001; accepted July 20, 2001 This paper is available online at http://molpharm.aspetjournals.org 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 electrophysio- logical and pharmacological properties were recorded from porcine GC by using whole-cell patch clamp: 1) a slowly acti- vating, noninactivating current (I Ks ) antagonized by clofilium, 293B, L-735,821, and L-768,673; and 2) an ultrarapidly activat- ing, slowly inactivating current (I Kur ) antagonized completely by clofilium and 4-aminopyridine and partially by tetraethylammo- nium, charybdotoxin, dendrotoxin, and kaliotoxin. The molec- ular identity of the K  channel genes underlying I Ks and I Kur was examined using reverse transcription-polymerase chain reac- tion 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, Kv1.3, and Kv2. Coimmunoprecipitation was used to establish the het- ero-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 - and -subunits was also documented, sug- gesting that the diverse electrophysiological and pharmacolog- ical properties of I Kur currents may reflect variation in the com- position and stoichiometry of the channel assemblies, as well as differences in post-translational modification of contributing Kv channel subunits. Our findings provide an essential back- ground 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 repre- sent either novel targets for assisted reproduction or potential sites of drug toxicity. Granulosa cells (GC) surround the oocyte within the ovar- ian follicle and play an essential role in creating the condi- tions required for follicular development, ovulation, fertiliza- tion, and implantation (Salustri et al., 1993). During folliculogenesis, GC undergo a series of mitotic divisions (pro- liferation) then acquire gonadotropin receptors and enhanced steroidogenic activity (differentiation). Autocrine-paracrine and endocrine regulation of the granulosa cell maturation have been extensively investigated and much is known about the specific roles of various growth factors, hormones, trans- membrane receptors, and second messengers (Steele and Leung, 1993). In contrast, the functional significance of volt- age-dependent ion channels in GC is far from understood. It has been reported that GC can generate action poten- tials (Mealing et al., 1994), and indirect evidence suggests that modulation of granulosa cell electrical activity may pro- vide a means to regulate cell function (Mattioli et al., 1990, 1991, 1993; Kusaka et al., 1993). For example, granulosa cell depolarization has been described as a consistent feature of oocyte maturation in different experimental systems (Mat- tioli et al., 1990). Voltage-gated potassium currents with distinct electrophysiological and pharmacological properties have been described in both acutely isolated and cultured GC, and have been shown to regulate granulosa cell resting These studies were supported by National Institutes of Heath grants HD34235 and HD36002 (to L.F.). D.E.M. and K.E.M. contributed equally to this study. ABBREVIATIONS: GC, granulosa cells; Kv1, voltage-gated K  channel subfamily 1 member; HRP, horseradish peroxidase; ECL, enhanced chemiluminescence; FSH, follicle-stimulating hormone; MK-499, [()-N-{1'-(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)- hydroxyspiro(2H-1-benzopyran-2,4'-piperidin)-6-yl]methanesulfonamide]; 293B, 4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethylchro- mane; LY97241, 4-ethyl-N-heptyl-4-nitrobenzenebutanamine ethanedioic acid; L735,821, (R)-2(ethylene-2,4-dichlorophenyl)-N-[2-oxo-5-phenyl- 1-methyl-2,3-dihydro-1H-benzol[e][1,4]diazepin-3-yl]acetamide; L768,673, (R)-2-(2,4-trifluoromethylphenyl)-N-[2-oxo-5-phenyl-1-(2,2,2- trifluoroethyl)-2,3-dihydro-1H-benzol[e][1,4]diazepin-3-yl]-acetamide; HEK, human embryonic kidney; HERG, human ether-a-go-go-related gene; PCR, polymerase chain reaction; DMEM, Dulbecco’s modified Eagle’s medium; I Ks , slow delayed rectifier K  current; I Kur , ultra-rapid delayed rectifier K  current; bp, base pair; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; PNGase-F, peptide-N-glycosidase F; 4-AP, 4-aminopyridine; TEA, tetraethylammonium; CTX, charybdotoxin; DTX, dendrotoxin; KTX, kaliotoxin; MTX, margatoxin; CHO, Chinese hamster ovary; RT, reverse transcription. 0026-895X/02/6101-201–213$3.00 MOLECULAR PHARMACOLOGY Vol. 61, No. 1 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 941/953855 Mol Pharmacol 61:201–213, 2002 Printed in U.S.A. 201 at ASPET Journals on May 5, 2017 molpharm.aspetjournals.org Downloaded from