Received for publication August 14, 2007.
Revised October 2, 2007.
Accepted for publication October 9, 2007.

Inhibition of TRPC5 channels by intracellular ATP

Abstract

TRPC5 channels are Ca²⁺-permeable non-selective cation channels activated by G-proteincoupled 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 G_q, almost abolished TRPC5 currents indicating that G_q 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 phosphorylationmediated effect. The activity of TRPC5 channels may be linked to cellular metabolism via changes in ATP levels, and could be involved in Ca²⁺ overload occurring after ischemia when ATP is depleted.

Key words: Muscarinic cholinergic, Ion channel regulation, Gq/11 family