Integrin-Dependent and -Independent Potentiation of L-type Calcium Current (Cav1.2) by Cell Stretch

Abstract

Stretch-induced (myogenic) contraction of vascular smooth muscle (VSM) requires calcium influx through L-type calcium channels (Cav1.2). Integrins play a role in this process because alpha5 beta1 and alphaVbeta3 integrin blocking antibodies prevent myogenic constriction. Recent studies in our lab indicate that Cav1.2 current is potentiated by alpha5beta1 integrin activation and requires phosphorylation by PKA and c-Src of Cav1.2 C-terminal residues. To test whether Cav1.2 channels are mechanosensitive and the possible role of integrins in this process, patch clamp methods were used to investigate the properties of either native or heterologously expressed Cav1.2 cannels by stretch of single cells plated onto a flexible substrate. Thin silicone membranes were coated with either fibronectin (FN) or poly-l-lysine (PLL) to assess integrin-dependent and -independent responses, respectively, and stretched using two blunt micropipettes driven in equal and opposite directions by piezoelectric translators. Graded stretch to 130% of resting cell length induced graded increases in Cav1.2 current (up to 63%) in HEK 293 cells expressing the neuronal channel isoform (Cav1.2c). The increase in current was ∼2-fold greater for cells adhering to FN than for cells on PLL. On FN, 130% longitudinal stretch of primary VSM cells induced ∼50% increases in Cav1.2 current. However, the magnitude of stretch-activated Cav1.2 current was the same on FN or PLL for cells expressing a Cav1.2 construct containing two C-terminal mutations (Y2122F/S1901A) to prevent phosphorylation by PKA and c-Src, or for cells expressing a Cav1.2 construct with the C-terminus truncated. Our results suggest that the Cav1.2 channel can be potentiated by membrane stretch, with one component due to intrinsic mechanosensitivity of the channel and a second component due to signaling through an integrin-dependent process.