Abstract
TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.
Highlights
Introduction of exogenousCaM did not overcome the inhibitory effect of high Ca2ϩ on the current amplitudes but still facilitated the rate of channel activation by receptor agonist
Consistent with this, we have shown that CaM increased the delay between the release of Ca2ϩ from internal storage compartments and the activation of Ca2ϩ influx via endogenous TRPC1 channels in Chinese hamster ovary (CHO) cells, which were subjected to the regulation by IP3R and CaM in a similar fashion as the exogenously expressed TRPC3 and TRPC4 in human embryonic kidney (HEK) 293 cells [12]
Ca2ϩ/CaM-mediated Facilitation of mouse TRPC5 (mTRPC5) Activation by Receptor Agonists—Stimulation of many cell surface receptors triggers intracellular Ca2ϩ signaling through activation of phospholipase C and breakdown of phosphoinositides
Summary
Introduction of exogenousCaM did not overcome the inhibitory effect of high Ca2ϩ on the current amplitudes but still facilitated the rate of channel activation by receptor agonist. We have compared the responses to thrombin and bradykinin (Bk) at concentrations that gave the maximal response (1 unit and 100 nM, respectively) in CHO cells stably expressing mTRPC5, and we obtained similar time constant values for half-activation of the outward current of 4.25 Ϯ 0.4 and 4.01 Ϯ 0.7 min, respectively.
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