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

Abstract

Previously we have described a constitutively active, Ca2+-permeable, non-selective cation channel in freshly dispersed rabbit ear artery myocytes which has similar properties to some of the canonical transient receptor potential (TRPC) channel proteins. In the present work we have compared the properties of constitutive channel activity with known properties of TRPC proteins by investigating the effect of selective anti-TRPC antibodies and pharmacological agents on whole-cell and single cation channel activity. Bath application of anti-TRPC3 antibodies markedly reduced channel activity in inside-out patches and also produced a pronounced reduction of both current amplitude and variance of constitutively active whole-cell cation currents whereas anti-TRPC1/4/5/6/7 antibodies had no effect on channel activity. In the presence of antigenic peptide, anti-TRPC3 antibodies had no effect on whole-cell or single cation channel activity. Bath application of flufenamic acid, Gd3+, La3+ and Ca2+ inhibited spontaneous channel activity in outside-out patches with IC50 values of 6.8 microm, 25 nm, 1.5 microm and 0.124 mm, respectively, which are similar values to those against TRPC3 proteins. Immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 proteins in ear artery myocytes, and these were predominately distributed at, or close to, the plasma membrane. These data provide strong evidence that native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 channel proteins and indicate that these proteins may have an important role in mediating this conductance.

Figure 1. Effect of anti-TRPC antibodies on constitutively active cation channel activity in inside-out patches

A and B, bath application of, respectively, anti-TRPC3a antibodies at 1 : 200 dilution and anti-hTRPC3 antibodies at 0.3 μg ml⁻¹ reversibly inhibited constitutively active channel activity in two different patches held at −50 mV. Insets show individual channel currents on a faster time scale. C, mean data showing the effect of anti-TRPC antibodies on constitutive channel activity represented as NP_o. The data using anti-TRPC antibodies from different sources have been pooled; note that the anti-TRPC3a antibody had no effect on channel activity following preincubation with its antigenic peptide (***P < 0.001).

Figure 2. Effect of anti-TRPC3 antibodies on constitutively active whole-cell cation currents

A, shows that inclusion of anti-TRPC3a antibodies at 1: 200 in the patch pipette solution markedly reduced both the amplitude (i) and variance (ii) of a constitutively active whole-cell cation current held at −50 mV. The inset shows individual current–voltage curves from Ai. B, shows that inclusion of anti-TRPC3a antibodies following preincubation with its antigenic peptide in the patch pipette solution had no effect on both the amplitude (i) or variance (ii) of the whole-cell current. The asterisk denotes when whole-cell configuration was obtained and the dotted lines represent zero holding current. C and D, mean data showing that anti-TRPC3a antibodies significantly reduced, respectively, holding current and current variance of constitutively active whole-cell cation currents (n = 7, **P < 0.01).

Figure 3. Effect of pharmacological agents on constitutively active cation channel currents in outside-out patches

A, shows that bath application of 10 μm flufenamic acid (FFA) reduced and 100 μm FFA completely blocked constitutive cation channel activity at −50 mV. B, C, D and E, respectively, show mean concentration–effect curves for FFA, Gd³⁺, La³⁺ and [Ca²⁺]_o on constitutive channel activity at −50 mV.

Figure 4. Immunocytochemical staining of rabbit ear artery myocytes for TRPC3 channel proteins

A shows a single confocal plane fluorescence image of a myocyte labelled with anti-TRPC3a antibodies (1 : 200) and B shows another myocyte labelled with anti-TRPC3a antibodies (1 : 200) preincubated with their antigenic peptide (1 : 100). Insets in A and B show transmitted light image of these myocytes. White circles in A indicate Regions 1 and 2, which were used to analyse the localization of fluorescence (see Methods and Supplemental material). A dotted line was used in B to outline the contour of a cell, due to its low fluorescence. C, mean data showing the localization of fluorescence related to binding of anti-TRPC3a antibodies. D, mean data showing intensity of fluorescence, expressed as average pixel fluorescence (intensity units per pixel). The values of all pixels in the confocal plane of the myocytes were added up and then divided by the number of pixels. The specificity of labelling was confirmed by greatly reduced fluorescence after preincubation with antigenic peptide or by virtual lack of fluorescence in the absence of primary antibodies (***P < 0.001).