Abstract
The Hydra Sodium Channel (HyNaC), a member of the DEG/ENaC ion channel family, is a peptide gated cation channel from the fresh water polyp Hydra magnipapillata. Four HyNaC subunits, HyNaC2 - HyNaC5, have already been characterized. It has been shown that HyNaCs 2, 3 and 5 assemble into a functional heterotrimeric channel, HyNaC2/3/5, which is activated with high affinity (EC50 ∼3.5 µM) by the endogenous neuropeptide Hydra-RFamide I. Here we report cloning of a new HyNaC subunit, HyNaC7. When expressed heterologously with HyNaC2 and 3, HyNaC7 increases apparent peptide affinity 20-40 times compared with HyNaC2/3/5 (EC50 ∼0.2 µM). Like HyNaC2/3/5, HyNaC2/3/7 is an unselective cation channel with a high calcium conductance and low apparent affinity for amiloride. By systematically swapping domains between HyNaC5 and HyNaC7 and functionally characterizing resulting chimeras, we identified the second transmembrane domain (TM2) and adjacent amino acids, located upstream as well as downstream of TM2, that determined the higher peptide affinity of HyNaC2/3/7. Currently, we investigate the influence of individual amino acids for ligand affinity. Together, our results show, that TM2 and C-terminal as well as N-terminal adjacent amino acids determine differences in ligand binding affinity of HyNaC2/3/7 and HyNaC2/3/5.
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