Cloning and expression of a FMRFamide-gated Na(+) channel from Helisoma trivolvis and comparison with the native neuronal channel.
We have cloned a cDNA encoding a Phe-Met-Arg-Phe-NH(2) (FMRFamide)-gated Na(+) channel from nervous tissue of the pond snail Helisoma trivolvis (HtFaNaC) and expressed the channel in Xenopus oocytes. The deduced amino acid sequence of the protein expressed by HtFaNaC is 65 % identical to that of the FMRFamide-gated channel cloned from Helix aspersa (HaFaNaC). HtFaNaC expressed in oocytes was less sensitive to FMRFamide (EC(50) = 70 microM) than HaFaNaC (EC(50) = 2 microM). The two had a similar selectivity for Na+. The amplitude of the FMRFamide response of HtFaNaC was increased by reducing the extracellular concentration of divalent cations. The conductance of the two channels was similar, but the mean open time of unitary events was shorter for expressed HtFaNaC compared to expressed HaFaNaC. Each channel was susceptible to peptide block by high agonist concentrations. In marked contrast to HaFaNaC and other amiloride-sensitive Na(+) channels, amiloride, and the related drugs benzamil and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), enhanced the FMRFamide response in oocytes expressing HtFaNaC cRNA. The potentiating effects of EIPA and benzamil were greater than those of amiloride. Unitary current analysis showed that with such drugs, there was channel blockade as well as an increased probability of channel opening. The similar permeability of the oocyte-expressed HtFaNaC and the Helisoma neuronal channel, and the susceptibility of both to agonist blockade and blockade by divalent cations, suggest that the channels are the same. However, neuronal channels were less susceptible to enhancement by amiloride analogues and in some patches were more sensitive to FMRFamide than expressed HtFaNaC.
Jeziorski
M C
MC
School of Biology, University of St Andrews, Fife KY16 9TS, UK.
Green
K A
KA
Sommerville
J
J
Cottrell
G A
GA
D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D019835:FMRFamide / Q000378:metabolism* / Q000494:pharmacology; D015870:Gene Expression; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics*; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017386:Sequence Homology, Amino Acid; D012908:Snails / Q000235:genetics* / Q000378:metabolism; D015222:Sodium Channels / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D014162:Transfection; D014981:Xenopus
C097095:FaNaCh protein, Helix aspersa; D018696:Neuroprotective Agents; D015222:Sodium Channels; C013407:benzamil; D019835:FMRFamide; D000584:Amiloride; C039614:ethylisopropylamiloride