title: Uncoupling of Cav1.2 from Ca(2+)-induced Ca(2+) release and SK channel regulation in pancreatic β-cells.
abstract: We investigated the role of Cav1.2 in pancreatic β-cell function by expressing a Cav1.2 II-III loop/green fluorescent protein fusion in INS-1 cells (Cav1.2/II-III cells) to disrupt channel-protein interactions. Neither block of KATP channels nor stimulation of membrane depolarization by tolbutamide was different in INS-1 cells compared with Cav1.2/II-III cells, but whole-cell Cav current density was significantly increased in Cav1.2/II-III cells. Tolbutamide (200 μM) stimulated insulin secretion and Ca(2+) transients in INS-1 cells, and Cav1.2/II-III cells were completely blocked by nicardipine (2 μM), but thapsigargin (1 μM) blocked tolbutamide-stimulated secretion and Ca(2+) transients only in INS-1 cells. Tolbutamide-stimulated endoplasmic reticulum [Ca(2+)] decrease was reduced in Cav1.2/II-III cells compared with INS-1 cells. However, Ca(2+) transients in both INS-1 cells and Cav1.2/II-III cells were significantly potentiated by 8-pCPT-2'-O-Me-cAMP (5 μM), FPL-64176 (0.5 μM), or replacement of extracellular Ca(2+) with Sr(2+). Glucose (10 mM) + glucagon-like peptide-1 (10 nM) stimulated discrete spikes in [Ca(2+)]i in the presence of verapamil at a higher frequency in INS-1 cells than in Cav1.2/II-II cells. Glucose (18 mM) stimulated more frequent action potentials in Cav1.2/II-III cells and primary rat β-cells expressing the Cav1.2/II-II loop than in control cells. Further, apamin (1 μM) increased glucose-stimulated action potential frequency in INS-1 cells, but not Cav1.2/II-III cells, suggesting that SK channels were not activated under these conditions in Cav1.2/II-III loop-expressing cells. We propose the II-III loop of Cav1.2 as a key molecular determinant that couples the channel to Ca(2+)-induced Ca(2+) release and activation of SK channels in pancreatic β-cells.
authors: Purdue University Life Sciences Graduate Program (R.E.J., E.P.S.P., A.M.L.) and Department of Medicinal Chemistry and Molecular Pharmacology (Y.W., M.L.G., A.E.S., I.M.S., G.H.H.), Purdue University, West Lafayette, Indiana 47907-2091.
authors: Yuchen
authors: Y
authors: Wang
authors: Rachel E
authors: RE
authors: Jarrard
authors: Evan P S
authors: EP
authors: Pratt
authors: Marcy L
authors: ML
authors: Guerra
authors: Amy E
authors: AE
authors: Salyer
authors: Allison M
authors: AM
authors: Lange
authors: Ian M
authors: IM
authors: Soderling
authors: Gregory H
authors: GH
authors: Hockerman
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000378:metabolism*; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D000242:Cyclic AMP / Q000031:analogs & derivatives; D004705:Endocytosis / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D039621:Eukaryotic Initiation Factor-3 / Q000378:metabolism; D052216:Glucagon-Like Peptide 1 / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism*; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D008297:Male; D017433:Protein Structure, Secondary; D051381:Rats; D017208:Rats, Wistar; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D014044:Tolbutamide / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology; D020703:ras GTPase-Activating Proteins / Q000378:metabolism
chemical_list: C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; D020746:Calcium Channels, L-Type; D039621:Eukaryotic Initiation Factor-3; C101203:IQ motif containing GTPase activating protein 1; D007328:Insulin; C419385:L-type calcium channel alpha(1C); D051640:Small-Conductance Calcium-Activated Potassium Channels; D020703:ras GTPase-Activating Proteins; D052216:Glucagon-Like Peptide 1; D014044:Tolbutamide; D014700:Verapamil; D000242:Cyclic AMP; D005947:Glucose; D002118:Calcium