| The non-selective cation channel TRPC5 is found in several regions of the mammalian central nervous system, including the hippocampus, cerebellum, amygdala and olfactory bulb. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous receptor stimulation, heterologously expressed TRPC5 currents are potentiated up to 25-fold. This potentiation is dependent upon calcium influx, as replacement of extracellular calcium with barium prevents it while addition of high extracellular calcium accelerates it. The site of action for calcium is intracellular, and simultaneous patch clamp and calcium imaging indicate that potentiation is triggered near 1 muM [Ca2+i]. Excised patch recordings confirm that potentiation consists of an increase in channel open probability with no alteration in conductance. TRPC5 calcium potentiation may support the detection of coincident receptor activation and intracellular calcium increases from other sources, or calcium permeating through the channel itself may generate a positive feedback loop in vivo. TRPC5 has been implicated in the negative regulation of growth cones, and we ask if the channel participates in the response to the repulsive guidance molecule semaphorin. Using a functional assay for growth cone collapse induced by semaphorin 3A, we show that knockout of TRPC5 results in a marked reduction in the effect of sema3A on cultured hippocampal neurons; inhibition of calpain, recently implicated in semaphorin signaling, produces the same result. However, calpain inhibition in TRPC5 knockout neurons does not further reduce the effects of sema3A, suggesting that calpain and TRPC5 may function in the same pathway. We find that both calpain-1 and calpain-2 activate TRPC5 in patch clamp studies and cleave the channel in vitro, generating a ∼850 amino acid fragment that retains ion channel function and conductivity. Calpain activation of TRPC5 may be a critical component of neuronal responses to negative guidance cues, with implications for neuronal development and axon regeneration following CNS injury. Finally, we show that the promiscuous proteases trypsin and papain activate TRPC4 and TRPC5, providing a useful tool for their study. |
