The Transient Receptor Potential Protein Homologue TRP6 Is the Essential Component of Vascular α 1 -Adrenoceptor–Activated Ca 2+ -Permeable Cation Channel

Abstract

—The Drosophila transient receptor potential protein (TRP) and its mammalian homologues are thought to be Ca²⁺-permeable cation channels activated by G protein (G_q/11)–coupled receptors and are regarded as an interesting molecular model for the Ca²⁺ entry mechanisms associated with stimulated phosphoinositide turnover and store depletion. However, there is little unequivocal evidence linking mammalian TRPs with particular native functions. In this study, we have found that heterologous expression of murine TRP6 in HEK293 cells reproduces almost exactly the essential biophysical and pharmacological properties of α₁-adrenoceptor–activated nonselective cation channels (α₁-AR–NSCC) previously identified in rabbit portal vein smooth muscle. Such properties include activation by diacylglycerol; S-shaped current-voltage relationship; high divalent cation permeability; unitary conductance of 25 to 30 pS and augmentation by flufenamate and Ca²⁺; and blockade by Cd²⁺, La³⁺, Gd³⁺, SK&F96365, and amiloride. Reverse transcriptase–polymerase chain reaction and confocal laser scanning microscopy using TRP6-specific primers and antisera revealed that the level of TRP6 mRNA expression was remarkably high in both murine and rabbit portal vein smooth muscles as compared with other TRP subtypes, and the immunoreactivity to TRP6 protein was localized near the sarcolemmal region of single rabbit portal vein myocytes. Furthermore, treatment of primary cultured portal vein myocytes with TRP6 antisense oligonucleotides resulted in marked inhibition of TRP6 protein immunoreactivity as well as selective suppression of α₁-adrenoceptor–activated, store depletion–independent cation current and Ba²⁺ influx. These results strongly indicate that TRP6 is the essential component of the α₁-AR–NSCC, which may serve as a store depletion–independent Ca²⁺ entry pathway during increased sympathetic activity.