TRPC3 Channel Blockers but not TRPC6 Blockers Inhibit Background Calcium Influx and Modulate Calcium Transients in Mouse Muscle Fibers

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We have recently shown that the cation channels TRPC3 and C6 are expressed in mouse skeletal muscle. Both mRNAs could be detected by RT-PCR and immunohistochemical staining revealed the presence of TRPC6 and in part TRPC3 in the sarcolemma of mouse muscle fibers. OAG, an activator of TRPC3/C6 and C7, stimulated background Ca2+ influx, supporting the hypothesis of functional expression of TRPC3 and/or C6 in skeletal muscle. TRPC6 could be pharmacologically activated, however, it does not seem to contribute to background Ca2+ influx, since the specific TRPC6 inhibitor ML-9 was ineffective. Here we studied whether the unspecific TRPC channel blocker 2-APB and/or the specific TRPC3 blocker Pyr3 affect muscular Ca2+ homeostasis. To investigate divalent cation influx we used single interosseus muscle fibers and applied the Mn2+ quench technique. Quench of Fura-2 fluorescence was recorded in the presence of 0.5 mM Mn2+ (excitation at 360 nm). Changes of cytoplasmic Ca2+ were measured using Fura-2 and alternate excitation at 340 and 380 nm. 2-APB inhibited background Ca2+ influx by more then 50% (n=44, p<0.01). The half time of decay of KCl induced calcium transients was as well significantly influenced by 2-APB (control vs. 2-APB; 3.61 ± 0.18 s vs. 3.18 ± 0.15 s; n= 27, 31; p < 0.05). The application of Pyr3 resulted in a marked inhibition of Fura-2 quench rate (control vs. Pyr3; 6.4 ±0.6 vs. 2.5±0.4 %/min; n=46, 46; p<0.01). We conclude that both channels, TRPC3 and TRPC6 are functional in the sarcolemma of isolated mouse muscle fibers. However, TRPC6 has no resting activity, but TRPC3 contributes substantially to the background Ca2+ influx of muscle fibers.