Spatial And Functional Coupling of The L‐Type Ca2+ Channel Cav1.2 with Ca2+‐Induced Ca2+ Release And cAMP Accumulation in INS‐1 cells

Exposure of pancreatic β‐cells to glucose generates concomitant oscillations in Ca2+ and cAMP which regulate insulin secretion, an essential function of β‐cells that promotes glucose homeostasis. Ca2+ influx through the L‐type Ca2+ channels Cav1.2 and Cav1.3 is further amplified by Ca2+ released from the ER via the ryanodine receptor (RyR) through a process called Ca2+‐induced Ca2+ release (CICR). L‐type Ca2+ 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 Cav1.2 II‐III loop, a highly divergent region between the amino acid sequences of Cav1.2 and Cav1.3 that is required for critical protein‐protein interactions, displaces endogenous Cav1.2 channels from lipid raft domains in INS‐1 cells (Cav1.2/II‐III cells). Given our finding that Cav1.2 colocalizes with the ER Ca2+ release channel RyR2 in INS‐1 cells, expression of the Cav1.2 II‐III loop may perturb proper spatial coupling of Cav1.2 with RyR. Consistent with this, we found that CICR is uncoupled from Cav1.2‐mediated Ca2+ influx in Cav1.2/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 Cav1.2/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 Cav1.2/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 Cav1.2/II–III cells. Further, both the L‐type Ca2+ channel blocker nicardipine (2 μM) and RyR inhibitor ryanodine (20 μM) completely abolished GS‐cAMP in control INS‐1 cells, suggesting that Ca2+ influx through L‐type Ca2+ channels and CICR are required for GS‐cAMP. Use of a plasma membrane‐targeted, Epac2‐based FRET sensor revealed that in Cav1.2/II–III cells, L‐type Ca2+ channel‐dependent GS‐cAMP is present at the plasma membrane. Taken together, Ca2+ influx through L‐type Ca2+ channels is sufficient to cause local GS‐cAMP; however, CICR largely amplifies the signal. Given our observation that GS‐cAMP is diminished in Cav1.2/II–III cells, we predicted that Cav1.2‐mediated CICR is preferentially coupled with GS‐cAMP. To test this, we utilized INS‐1 cells stably expressing dihydropyridine‐insensitive (DHPi) Cav1.2 and Cav1.3 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 Cav1.2/DHPi and Cav1.3/DHPi cells. Surprisingly, isradipine significantly but incompletely inhibited GS‐cAMP in both cell lines, whereas diltiazem completely abolished the responses. Thus, Cav1.2 or Cav1.3 can sustain GS‐cAMP in the absence of the corresponding channel. Overall, we conclude that both Ca2+ influx through L‐type Ca2+ channels and CICR are required for efficient GS‐cAMP, and both Cav1.2 and Cav1.3 are involved in this process.Support or Funding InformationThis 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).