Modulation of the FMRFamide-gated Na channel by external Ca.

FMRFamide-gated Na channel (FaNaC) is a member of the DEG/ENaC family. Amino acid sequence of the second transmembrane region (TM2) of FaNaC is quite similar to that of the acid-sensing ion channels (ASIC) of the same family. In the upper part of TM2, there are two aspartate residues (D552 and D556 in Aplysia FaNaC, AkFaNaC) which construct two negative rings in the external vestibule. In the present study, we examined the function of D552/D556 mutants of AkFaNaC in Xenopus oocytes with special interest in Ca sensitivity of FaNaC. The FMRFamide-evoked current through AkFaNaC was depressed by submillimolar Ca such that the current in Ca-free condition was 2-3-fold larger than that in the control solution which contained 1.8 mM CaCl . Both D552 and D556 were found to be indispensable for the sensitivity of FaNaC to submillimolar Ca. Unexpectedly, however, both acidic residues were not essential for the inhibition by millimolar Ca concentrations. The Ca-sensitive gating of FaNaC was recapitulated by an allosteric model in which Ca-bound channels are more difficult to open. The desensitization of FaNaC was also inhibited by Ca, which was abolished in some D552/D556 mutants. Structural models of FaNaC made by homology modeling showed that the distance between oxygen atoms of D552 and D556 on the adjacent subunits is close enough to coordinate Ca in the nonconducting desensitized channel but not in the open channel. The results suggest that Ca coordination between oxygen atoms of D552 and D556 disturbs the opening transition as well as the desensitization of FaNaC.