Figure 6

Formation of a TRPC3/Nox2 complex promotes TRPC3 channel activity through stabilization at the plasma membrane.

(a) Effect of Nox2 siRNA on expression of TRPC3 in NRCMs (n = 3). (b) Representative images showing the levels of TRPC3-GFP and GFP expression in HEK293 cells co-expressing p22^phox or Nox2 (n = 3). (c) Expression of TRPC3-GFP mRNA in HEK293 cells co-expressing p22^phox or Nox2 (n = 3). (d–f) Representative time courses of TRPC3 currents (d) and the current-voltage (I-V) relationships (e) and peak TRPC3 current densities at −60 mV (f) induced by 100 μM carbachol (CCh) in HEK293 cells expressing TRPC3-mCherry alone or with p22^phox, Nox2, both p22^phox and Nox2, or Nox2 treated with DPI. DPI (0.3 μM) was treated 1 min before CCh stimulation. (g,h) Representative Ca²⁺ responses in the presence (g) or absence (h) of pyrazole-3 (Pyr3, 1 μM) upon mechanical stretch (MS) application. (i) Peak Ca²⁺ increases after MS in NRCMs treated with (n = 61) or without Pyr3 (n = 78). (j) Changes of minimal [Ca²⁺]_i before and after MS application. Minimal [Ca²⁺]_i from Ca²⁺ responses in every 1 min were analyzed and represented as diastolic [Ca²⁺]_i. (k) Schematic images showing phosphorylation of p47^phox via TRPC3-PKCβ activation induced by MS in the heart. (l–n) Effects of TRPC3 (l,m) or PKCβ (n); 10 μM Gö6976) inhibitors on p47^phox phosphorylation induced by MS in NRCMs (n = 3). (o) MS-induced ROS generation in NRCMs treated with a PKCβ inhibitor (n = 3). (p) Co-immunoprecipitation of TRPC3 with PKCβ, Nox2 and p22^phox in mouse hearts 1week after TAC operation (n = 3). Error bars, s.e.m.*P < 0.05, **P < 0.01.