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 Ca(2+)-permeable cation channels activated by G protein (G(q/11))-coupled receptors and are regarded as an interesting molecular model for the Ca(2+) 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 alpha(1)-adrenoceptor-activated nonselective cation channels (alpha(1)-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 Ca(2+); and blockade by Cd(2+), La(3+), Gd(3+), 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 alpha(1)-adrenoceptor-activated, store depletion-independent cation current and Ba(2+) influx. These results strongly indicate that TRP6 is the essential component of the alpha(1)-AR-NSCC, which may serve as a store depletion-independent Ca(2+) entry pathway during increased sympathetic activity.

Intrinsic light responses of retinal ganglion cells projecting to the circadian system

“…Clapham et al, 2001;Minke & Cook, 2002). Specifically, the I-V characteristics closely resemble those reported for the TRPC6 channel in A7r5 smooth muscle cells (Jung et al, 2002), the mTRP6 channel in vascular smooth muscle (Inoue et al, 2001), and the TRPC4 and TRPC5 channels expressed in embryonic kidney cells (Shaefer et al, 2000). Furthermore, the I-V characteristics and amplitude of the light-activated current at −60 mV were largely unaffected by total replacement of extracellular Na + with choline.…”

Rho kinase is involved in Ca2+ entry of rat penile small arteries

“…Several members of the canonical transient receptor potential channel (TRPC) family act as Ca 2ϩ -permeable cation channels that can be activated in response to stimulation of G protein (G q/11 )-coupled receptors (8,12). TRPC6 is highly expressed in vascular smooth muscle cells and has been suggested to be the molecular correlate of the ␣ 1 -adrenoceptoractivated nonselective cation channel in rabbit portal vein (1,12) and mesenteric artery (11) myocytes.…”

“…TRPC6 is highly expressed in vascular smooth muscle cells and has been suggested to be the molecular correlate of the ␣ 1 -adrenoceptoractivated nonselective cation channel in rabbit portal vein (1,12) and mesenteric artery (11) myocytes. Moreover, TRPC6 and one of its splicing variant (TRPC6␣) are expressed in corporal myocytes from human CC (27), and, therefore, this TRPC subtype could be a molecular candidate for the nonselective cation channel activated by ␣ 1 -adrenoceptors and regulated by Rho kinase in penile arteries.…”

“…In some experiments, Ba 2ϩ (1 mM BaCl2) was used instead of Ca 2ϩ to evaluate the influx of divalent cations activated by Phe (9,12). The extent of Ba 2ϩ influx was assessed as the ratio of fluorescence intensity at 340 and 380 nm after correction for autofluorescence in fura-2 AM-loaded penile arteries.…”

“…The contribution of nonselective cation channels to the Ca 2ϩ entry stimulated by ␣ 1 -adrenoreceptor activation was specifically evaluated using Ba 2ϩ fluorescence instead of Ca 2ϩ to investigate the influx of divalent cations activated by Phe without any intervention of Ca 2ϩ extrusion or Ca 2ϩ storage mechanisms (12). Dorsal penile arteries were treated with nifedipine (1 M) to block VOC channels in a nominally Ca 2ϩ -free solution and stimulated with Phe (3 M).…”