Ca²⁺–calmodulin‐dependent myosin light chain kinase is essential for activation of TRPC5 channels expressed in HEK293 cells

Abstract

Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca2+ influx in vertebrate cells. We previously reported the involvement of intracellular Ca 2+ in the receptor-mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca 2+ , and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca 2+ entry through TRPC5 channels, induced upon stimulation of the G-protein-coupled ATP receptor, was abolished by treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, inhibitors of Ca 2+ -calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML-9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole-cell patch-clamp technique was inhibited by ML-9, whereas TRPC5 channel activity observed in the cell-excised, inside-out patch was unaffected by ML-9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML-9 in HEK293 cells. These findings strongly suggest that intracellular Ca 2+ -calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.

Elucidation of a TRPC6-TRPC5 Channel Cascade That Restricts Endothelial Cell Movement

“…We postulate that the rise in [Ca 2ϩ ] i through TRPC6 channels activates TRPC5. Increased [Ca 2ϩ ] i can induce calcium-dependent signaling events such as myosin light-chain kinase activation that can activate TRPC5 (Shimizu et al, 2006). The role of myosin light-chain kinase in lysoPC-induced activation of TRPC5 is currently under investigation in our laboratory.…”

Potentiation of TRPC5 by Protons

“…Spontaneous currents are characteristic for the murine clone (10,13,23) and are dependent on Ca 2ϩ possibly acting via calmodulin and myosin light chain kinase (24). A pH reduction from 7.4 to 6.5 indeed led to robust inward currents between Ϫ50 and Ϫ800 pA (Ϫ27.3 Ϯ 5.3 pA/pF, n ϭ 21) at a holding potential of Ϫ50 mV in HEK 293 cells transfected with the channel cDNA.…”

Mechanism of Ca2+-dependent desensitization in TRP channels

“…38 In contrast, phosphorylation by protein kinase A and protein kinase C has been proposed to slow desensitization in TRPV1 channels, 39,40 and protein kinase C has also been implicated in reversing desensitization of TRPM4 channels. 41 Ca 2+ /CaM regulated phosphorylation/dephosphorylation has also been proposed: phosphorylation of TRPC5 by myosin light chain kinase is associated with slowing of desensitization 42 and dephosphorylation of TRPV1 by calcineurin is reported to promote desensitization. 40,43 Thus, although phosphorylation seems to be involved in activation, and perhaps desensitization, of several TRP channels, no unifying theme has yet emerged.…”