TRPC3 properties of a native constitutively active Ca2+‐permeable cation channel in rabbit ear artery myocytes

Abstract

We have previously shown a constitutively active and noradrenaline‐evoked Ca2+‐permeable cation channel in rabbit ear artery myocytes which is proposed to have an important role is setting resting membrane conductance and increasing excitability of vascular tissue. In the present work we compared the properties of these native constitutively active cation channels with known properties of canonical transient receptor potential (TRPC) cation channel proteins at the single channel level using freshly dispersed rabbit ear artery myocytes and patch clamp techniques. Bath application of two anti‐TRPC3 antibodies raised against distinct epitopes on the TRPC3 channel protein markedly reduced constitutive channel activity in inside‐out patches whereas antibodies raised TRPC1,4,5,6 and 7 channel proteins had no effect on channel activity. Moreover in control experiments bath application of anti‐TRPC3 antibodies preincubated with its antigenic peptide also had no effect on channel activity. Bath application of flufenamic acid, Gd3+, La3+ and Ca2+ inhibited spontaneous channel activity in outside‐out patches with IC50 values similar to those previous described for expressed TRPC3 channel proteins in cell lines. In addition immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 channel proteins at, or close to, the plasma membrane. These data provide strong evidence that the native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 cation channel proteins and indicate that these proteins may have an important role in mediating this conductance.

Knockdown of TRPC3 with siRNA coupled to carbon nanotubes results in decreased insulin‐mediated glucose uptake in adult skeletal muscle cells

“…Excitation was at 488 and 568 nm, and the emitted light was collected through 522-and 605-nm narrowband filters. Previous control experiments showed no staining with the anti-TRPC3 antibody when muscle cells were exposed to an excess of TRPC3 blocking peptide (24,26), whereas exposure to an excess of TRPC6 blocking peptide had no effect on the TRPC3 staining (25).…”

“…Figure 1A shows that application of OAG (30 M) induced a current (I OAG ) with an almost linear currentvoltage relation. I OAG was almost completely blocked with anti-TRPC3 antibody in the patch pipette, indicating that the current is conducted via TRPC3 (25,26). I OAG was also inhibited by the pharmacological agents 2-aminoethoxydiphenyl borate (2-APB; 100 M) and Gd 3ϩ (1 M), which are frequently used to inhibit TRPC channel activity (Fig.…”

“…Therefore, we focus on TRPC3 in the present study and test the hypothesis that TRPC3 channels are involved in insulin-mediated glucose uptake in adult skeletal muscle. In an initial set of experiments, the OAG-induced NSCC was measured in skeletal muscle cells with anti-TRPC3 antibodies in the patch pipette, which previously have been shown to inhibit the NSCC mediated through TRPC3 channels (25,26). In a second set of experiments, TRPC3 protein expression was knocked down in adult skeletal muscle using small-interfering RNA (siRNA) linked to functionalized carbon nanotubes (CNTs).…”

Multiple activation mechanisms of store‐operated TRPC channels in smooth muscle cells

“… —, information not available; CIF, calcium influx factor; iPLA 2 , calcium‐independent phospholipase A 2 ; LPLs, lysophospholipids; PKC, protein kinase C; CaM, calmodulin. a Trepakova et al (2001); b Smani et al (2004); c Albert & Large (2002 a ); d Albert & Large (2002 b ); e Albert et al (2006 b ); f authors' unpublished data, see Fig. 4; g Saleh et al (2006); h Golovina et al (2001); i Ma et al (2000); j Ma et al (2002); k Wang et al (2004); l Parekh & Putney (2005); m Lewis, 2007).…”

TRPC1 proteins confer PKC and phosphoinositol activation on native heteromeric TRPC1/C5 channels in vascular smooth muscle: comparative study of wild‐type and TRPC1 −/− mice

“…Homomeric TRPC5 channels are expressed in TRPC1 ؊/؊ VSMCs CPA stimulates channel activity in TRPC1 Ϫ/Ϫ VSMCs, which has three conductance states of 14 pS, 32 pS, and 53 pS. These conductance levels are likely to represent subconductance states as the conductance values are not multiples of the lowest conductance level, the three open levels are observed in all patches and at low open probabilities, all three conductance levels had similar E r , and multiple subconductance states seem to be a characteristic of native TRPC channels in VSMCs, which do not contain TRPC1 (8,33,34).…”