Regulation of TRPC5 Channels by Intracellular ATP

Abstract

TRPC5 channels are Ca2+-permeable non-selective cation channels activated by G-protein-coupled receptors, although the mechanisms responsible for channel activation and regulation are poorly understood. Carbachol-activated TRPC5 currents were recorded by the whole-cell patch clamp technique from HEK-293 cells transiently transfected with TRPC5 and the M1 muscarinic receptor. Some published studies of TRPC5 currents have included ATP and/or GTP in the patch pipette, whereas others used an ATP- and GTP-free pipette solution. We initially included these two nucleotides in the patch pipette but found that TRPC5 currents were absent or were very small. Recordings made with an ATP- and GTP-free pipette solution produced large and robust TRPC5 currents. Under these conditions, treatment of cells with Pasteurella multocida toxin (PMT), a selective inhibitor of Gαq, almost abolished TRPC5 currents indicating that Gαq is necessary for activation of TRPC5 by the M1 receptor. To study the effect of intracellular ATP on TRPC5 channels, an intracellular perfusion system was used. Perfusion of ADP or control pipette solution had no effect, whereas perfusion of ATP or AMP-PNP, a non-hydrolysable analog of ATP, significantly inhibited TRPC5 currents. Thus, the effects of ATP have structural specificity and probably involve a direct effect on the channel rather than a phosphorylation-mediated effect.

To study the effect of ATP on TRPC5 channels in more detail, single-channel recordings in the inside-out patch configuration were used. With intracellular Ca2+ buffered to 100 nM, application of 4 mM ATP to the intracellular face of TRPC5 channels dramatically but reversibly reduced channel activity at both positive and negative potentials without affecting the unitary current amplitude or the dwell time of the channel, suggesting that ATP may bind to the channel protein and may affect the ability of the channel to open or to remain in an open, non-desensitized state. In other experiments where the effects of 2 mM ATP were studied, we used 0 nM intracellular Ca2+ instead of 100 nM Ca2+ to stabilize the membranes of patches to allow for prolonged recordings from the patches. In recordings with nominally 0 nM intracellular Ca2+ the amplitude of unitary TRPC5 currents was reduced at positive potentials, but unaffected at negative potentials, as compared to the amplitude of TRPC5 currents recorded with 100 nM intracellular Ca2+. At positive potentials, application of 2 mM ATP dramatically and reversibly decreased TRPC5 channel activity without affecting the dwell time of the channel or the amplitude of TRPC5 currents. At negative potentials however, application of 2 mM ATP with 0 nM intracellular Ca2+ increased TRPC5 channel activity, suggesting a potential interaction between Ca2+, ATP, and TRPC5 channels.

Taken together, our data suggest that the activity of TRPC5 channels may be linked to cellular metabolism via changes in ATP levels, and that TRPC5 channel activity could be involved in Ca2+ overload occurring after ischemia when ATP is depleted.