Dual Regulation of R-Type Ca(V)2.3 Channels by M(1) Muscarinic Receptors

Voltage-gated Ca(2+) (Ca(V)) channels are dynamically modulated by G protein-coupled receptors (GPCR). The M(1) muscarinic receptor stimulation is known to enhance Ca(V)2.3 channel gating through the activation of protein kinase C (PKC). Here, we found that M(1) receptors also inhibit Ca(V)2.3 currents when the channels are fully activated by PKC. In whole-cell configuration, the application of phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated Ca(V)2.3 currents by ∼two-fold. After the PMA-induced potentiation, stimulation of M(1) receptors decreased the Ca(V)2.3 currents by 52 ± 8%. We examined whether the depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is responsible for the muscarinic suppression of Ca(V)2.3 currents by using two methods: the Danio rerio voltage-sensing phosphatase (Dr-VSP) system and the rapamycin-induced translocatable pseudojanin (PJ) system. First, dephosphorylation of PI(4,5)P(2) to phosphatidylinositol 4-phosphate (PI(4)P) by Dr-VSP significantly suppressed Ca(V)2.3 currents, by 53 ± 3%. Next, dephosphorylation of both PI(4)P and PI(4,5)P(2) to PI by PJ translocation further decreased the current by up to 66 ± 3%. The results suggest that Ca(V)2.3 currents are modulated by the M(1) receptor in a dual mode—that is, potentiation through the activation of PKC and suppression by the depletion of membrane PI(4,5)P(2). Our results also suggest that there is rapid turnover between PI(4)P and PI(4,5)P(2) in the plasma membrane.