Spatial And Functional Coupling of The L‐Type Ca 2+ Channel Ca v 1.2 with Ca 2+ ‐Induced Ca 2+ Release And cAMP Accumulation in INS‐1 cells

Exposure of pancreatic β‐cells to glucose generates concomitant oscillations in Ca 2+ and cAMP which regulate insulin secretion, an essential function of β‐cells that promotes glucose homeostasis. Ca 2+ influx through the L‐type Ca 2+ channels Ca v 1.2 and Ca v 1.3 is further amplified by Ca 2+ released from the ER via the ryanodine receptor (RyR) through a process called Ca 2+ ‐induced Ca 2+ release (CICR). L‐type Ca 2+ channel blockers abolish glucose‐stimulated cAMP accumulation (GS‐cAMP); however, the role of CICR in this process is not well understood. We've demonstrated that expression of the Ca v 1.2 II‐III loop, a highly divergent region between the amino acid sequences of Ca v 1.2 and Ca v 1.3 that is required for critical protein‐protein interactions, displaces endogenous Ca v 1.2 channels from lipid raft domains in INS‐1 cells (Ca v 1.2/II‐III cells). Given our finding that Ca v 1.2 colocalizes with the ER Ca 2+ release channel RyR2 in INS‐1 cells, expression of the Ca v 1.2 II‐III loop may perturb proper spatial coupling of Ca v 1.2 with RyR. Consistent with this, we found that CICR is uncoupled from Ca v 1.2‐mediated Ca 2+ influx in Ca v 1.2/II‐III cells. To examine the role of CICR in GS‐cAMP, we expressed the Epac1‐based, cytosolic FRET sensor H187 in control INS‐1 and CICR‐deficient Ca v 1.2/II–III cells. When cAMP production was stimulated with the adenylyl cyclase activator forskolin, a robust response was detected in both control INS‐1 and Ca v 1.2/II–III cells, and there was no significant difference between them. In response to glucose (18 mM), we detected cAMP accumulation above baseline in control INS‐1 cells; however, glucose failed to elicit a detectable increase in Ca v 1.2/II–III cells. Further, both the L‐type Ca 2+ channel blocker nicardipine (2 μM) and RyR inhibitor ryanodine (20 μM) completely abolished GS‐cAMP in control INS‐1 cells, suggesting that Ca 2+ influx through L‐type Ca 2+ channels and CICR are required for GS‐cAMP. Use of a plasma membrane‐targeted, Epac2‐based FRET sensor revealed that in Ca v 1.2/II–III cells, L‐type Ca 2+ channel‐dependent GS‐cAMP is present at the plasma membrane. Taken together, Ca 2+ influx through L‐type Ca 2+ channels is sufficient to cause local GS‐cAMP; however, CICR largely amplifies the signal. Given our observation that GS‐cAMP is diminished in Ca v 1.2/II–III cells, we predicted that Ca v 1.2‐mediated CICR is preferentially coupled with GS‐cAMP. To test this, we utilized INS‐1 cells stably expressing dihydropyridine‐insensitive (DHPi) Ca v 1.2 and Ca v 1.3 channels. Here, the introduced DHPi channel is resistant to the DHP isradipine (2 μM); however, it remains sensitive to diltiazem (500 μM). As expected, we found that glucose markedly stimulated cAMP accumulation in both Ca v 1.2/DHPi and Ca v 1.3/DHPi cells. Surprisingly, isradipine significantly but incompletely inhibited GS‐cAMP in both cell lines, whereas diltiazem completely abolished the responses. Thus, Ca v 1.2 or Ca v 1.3 can sustain GS‐cAMP in the absence of the corresponding channel. Overall, we conclude that both Ca 2+ influx through L‐type Ca 2+ channels and CICR are required for efficient GS‐cAMP, and both Ca v 1.2 and Ca v 1.3 are involved in this process. Support or Funding Information This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Disease (R01 DK064736) (to Gregory Hockerman) and a Purdue Research Foundation Grant (to Evan Pratt and Gregory Hockerman).