The L-type voltage-gated calcium channels: The role of Cav1.2 in pancreatic B cells and new perspective on the molecular pharmacology of Cav1.3

Yuchen Wang, Purdue University

Abstract

Ca2+ influx through L-type voltage-gated calcium channels (L-VGCCs), Cav1.2 and Cav1.3, plays a crucial role in a variety of important physiological processes such as hormone release and muscle contraction. However, due to the high homology (>70%) shared between the two subtypes and lack of a subtype-specific L-VGCC blocker, the distinct roles of Cav1.2 and Cav1.3 in these important processes still remain unknown. The intracellular loop connecting domains II and III (II-III loop) of Cav1.2 and Cav1.3 is one of the most divergent regions where only about 40% amino acid identity is shared between the two. Our previous findings suggest that the Cav1.2/II-III loop plays a critical role in positioning Cav1.2 in signaling complexes which regulate different Ca2+-dependent downstream signaling events To investigate the role of the Cav1.2/II-III loop in β cells as well as the functional consequences caused by the observed Cav1.2 dislocation out of lipid rafts in INS-1 cells stably overexpressing the Cav1.2/II-III loop (Cav1.2/II-III cells), I studied the Ca2+ mobilization and insulin secretion stimulated with sulfonylureas and glucose in both INS-1 and Cav1.2/II-III cells. I measured the insulin secretion stimulated by tolbutamide and gliclazide for both cell lines and found that the Ca2+ release from the internal stores contributes a significant portion to sulfonylurea-stimulated insulin secretion in INS-1 cells but does not contribute to the secretion in Cav1.2/II-III cells. I also measured changes in intracellular Ca2+ level stimulated by glucose+GLP-1+verapamil using single cell live imaging and showed that Ca2+-induced Ca2+ release is disrupted in Cav1.2/II-III cells. The small-conductance potassium (SK) channels are activated by Ca2+ and modulate the action potential frequency in pancreatic β cells and neurons. However, the Ca2+ source activating SK channels still remains unknown. We previously showed that SK channels are not activated in Cav1.2/II-III cells in response to 18 mM glucose. Here, I studied the activity of the endogenous SK channels by measuring apamin-sensitive K+ current under 0 and 2 μM intracellular free Ca2+ ([Ca2+]i) in both cell lines. The SK selective blocker apamin did not block K+ current at 0 μM [Ca2+]i but achieved similar current fraction block in both INS-1 and Cav1.2/II-III cells at 2 μM [Ca2+]i. Analysis of current density showed no significant difference between the two cell lines, suggesting that the disrupted CICR in Cav1.2/II-III cells might contribute to the lack of SK activation observed in these cells upon glucose stimulation. I also investigated the molecular pharmacology of Cav1.3 by studying the effect of the transmembrane IIIS5 region and the extracellular IIIS5-IIIP region on Cav1.3’s sensitivity to dihydropyridines (DHPs) and calcicludine. The two amino acids (Thr 1033 and Glu 1037) in IIIS5 of Cav1.3 were mutated to Tyr and Met, respectively, and the IC50 of nifedipine blocking the mutant channel increased by ~200 fold with the IC50 of a non-DHP diltiazem not significantly changed. To study the role of the extracellular IIIS5-IIIP region, a chimeric Cav1.3+ was created by swapping the IIIS5-IIIP of Cav1.2 into the backbone of Cav1.3. The sensitivity of Cav1.2, Cav1.3 and Cav1.3+ to nifedipine and calcicludine block was compared. Calcicludine selectively blocked Cav1.2 over Cav1.3 at three different concentrations, but Cav1.3+ displayed a calcicludine sensitivity comparable to that of Cav1.2. Cav1.2 is slightly but significantly more sensitive to nifedipine and the swapped IIIS5-IIIP region from Cav1.2 increased nifedipine block of Cav1.3+ by ~ 4 fold compared to parent Cav1.3.

Degree

Ph.D.

Advisors

Hockerman, Purdue University.

Subject Area

Molecular biology|Cellular biology|Pharmacology

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