Abstract
BACKGROUND: TRPC5 proteins form plasma membrane cation channels and are expressed in the nervous and cardiovascular systems. TRPC5 activation leads to cell depolarization and increases neuronal excitability, whereas a homologous TRPC1 inhibits TRPC5 function via heteromerization. The mechanism underlying the inhibitory effect of TRPC1 in TRPC5/TRPC1 heteromers remains unknown. METHODS: We used electrophysiological techniques to examine the roles of subunit stoichiometry and positively charged luminal residues of TRPC1 on TRPC5/TRPC1 function. We also performed molecular dynamics simulations. RESULTS: We found that increasing the relative amount of TRPC1 in TRPC5/TRPC1 heteromers reduced histamine-induced cation influx through the heteromeric channels. Consistently, histamine-induced cation influx was small in cells co-expressing TRPC5-TRPC1 concatemers and TRPC1, and large in cells co-expressing TRPC5-TRPC1 concatemers and TRPC5. Molecular dynamics simulations revealed that the TRPC1 protein has two positively charged lysine residues that are facing the heteromeric channel pore lumen. Substitution of these lysines with asparagines decreased TRPC1's inhibitory effect on TRPC5/TRPC1 function, indicating that these lysines may regulate cation influx through TRPC5/TRPC1 heteromers. Additionally, we established that extracellular Mg(2+) inhibits cation influx through TRPC5/TRPC1, contributing to channel regulation. CONCLUSIONS: We revealed that the inhibitory effect of TRPC1 on heteromeric TRPC5/TRPC1 function likely involves luminal lysines of TRPC1.