Investigating the role of L-type voltage-gated calcium channels in electrical events in pancreatic Beta cells
Abstract
L-type voltage-gated calcium channels play a central role in providing the influx of calcium that stimulates electrical activity and insulin secretion in pancreatic β cells. Cav1.2 and Cav1.3 are the two L-type channels that are present in β cells and insulin-secreting cell lines; however the defined role that each channel has in contributing to the electrical activity that drives insulin secretion remains to be fully understood. Cav1.2 and Cav1.3 share much similarity in amino acid sequence, yet there is a higher sequence divergence in the intracellular loops that connect the homologous transmembrane domains of the α 1 pore-forming subunit. The intracellular loop connecting domains II and III (II-III loop) of Cav1.2 and Cav1.3 share only about 40% amino acid sequence identity, making this region one of the most divergent regions of the channel. The II-III loop region of voltage-gated calcium channels has been highly studied regarding its function as a site for protein-protein interactions which positions the calcium channels in signaling complexes that modulate neurotransmitter and hormone release. To investigate the role of the Cav II-III loop in β cells, we stably expressed the II-III loop portion of each channel in INS-1 pancreatic β cells and created stable INS-1 cell lines called Cav1.2/II-III cells (II-III loop of Cav1.2 expressed) and Cav1.3/II-III cells (II-III loop of Cav1.3 expressed). We have previously reported that Cav1.2 and Cav1.3 are localized to lipid raft regions and that expressing the II-III loop peptide of either channel specifically displaces the respective channel out of lipid rafts. Displacement of Ca v1.2 or Cav1.3 could have functional consequences for the electrical activity that initiates and regulates insulin secretion. I measured whole-cell voltage-gated calcium channel activity and K ATP channel activity in INS-1, Cav1.2/II-III, and Ca v1.3/II-III cells to determine if there was any effect of channel displacement from lipid rafts on ion channel activity. I examined the current-voltage relationship of voltage-gated calcium channels and observed no difference in the three cell lines, suggesting that the endogenous channels are still activated in their normal membrane potential threshold range. In the Cav1.2/II-III cells, the whole-cell IBa density was actually significantly greater compared to INS-1 and Cav1.3/II-III cells. I measured KATP channel activity and modulation by diazoxide and sulfonylureas in each cell line. I did not detect a significant difference in tolbutamide sensitivity of KATP channel currents or gliclazide sensitivity of KATP channel-mediated membrane depolarization. Displacement of Cav 1.2 or Cav1.3 does not diminish voltage-gated calcium channel activity or affect the function of the KATP channel. I used the perforated-patch clamp recording configuration to measure glucose induced depolarization and action potentials in INS-1, Cav1.2/II-III, and Cav1.3/II-III cells to determine if there was an effect of Cav1.2 or Cav1.3 being displaced from lipid rafts. I determined that there was no significant difference in the level of depolarization induced by 18 mM glucose in each cell line, supporting my previous observations that the activity of voltage-gated calcium channels and the KATP channel in these cells has not been compromised. I observed that displacement of Cav1.2 or Cav1.3 affected action potential spiking in the Cav1.2/II-III, and Cav1.3/II-III cells. There was an increase in frequency of action potentials in the Cav1.2/II-III cells compared to INS-1 cells, suggesting Cav1.2 is integral for contributing to the Ca2+ signal that leads to SK channel activation for regulation of action potential frequency. I also observed a decrease in frequency of action potentials in the Cav1.3/II-III cells compared to the INS-1 cells, suggesting Cav1.3 is integral for contributing to the Ca2+ signal that is required for induction of action potentials. These observations provide evidence for coupling of Cav1.2 or Cav1.3 to mechanisms of action potential regulation in pancreatic β cells.
Degree
Ph.D.
Advisors
Hockerman, Purdue University.
Subject Area
Molecular biology|Endocrinology|Pharmacology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.