Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation.
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.
Ordaz
Benito
B
Departamento de Biología Celular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, México City, DF 04510, México.
Tang
Jisen
J
Xiao
Rui
R
Salgado
Alfonso
A
Sampieri
Alicia
A
Zhu
Michael X
MX
Vaca
Luis
L
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015290:Second Messenger Systems / Q000502:physiology; D050052:TRPC Cation Channels
D015220:Calcium Channels; D002147:Calmodulin; D027682:Cation Transport Proteins; D050052:TRPC Cation Channels; C112021:Trpc5 protein, mouse; D002118:Calcium