Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel

Abstract

The peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) and structurally related peptides are present both in invertebrate and vertebrate nervous systems. Although they constitute a major class of invertebrate peptide neurotransmitters, the molecular structure of their receptors has not yet been identified. In neurons of the snail Helix aspersa, as well as in Aplysia bursting and motor neurons, FMRFamide induces a fast excitatory depolarizing response due to direct activation of an amiloride-sensitive Na+ channel. We have now isolated a complementary DNA from Helix nervous tissue; when expressed in Xenopus oocytes, it encodes an FMRFamide-activated Na+ channel (FaNaCh) that can be blocked by amiloride. The corresponding protein shares a very low sequence identity with the previously cloned epithelial Na+ channel subunits and Caenorhabditis elegans degenerins, but it displays the same overall structural organization. To our knowledge, this is the first characterization of a peptide-gated ionotropic receptor.

DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature

“…This ligand-gated DEG͞ENaC channel activates and desensitizes in the continued presence of the peptide FMRFamide (28). In contrast to acid-gated channels, desensitization of FaNaCh showed little temperature dependence (Q 10 ϭ 1.3, Fig.…”

Block of the Helix FMRFamide‐gated Na+ channel by FMRFamide and its analogues

“…As previously noted, a characteristic of the FaNaC expressed in oocytes is the occasional presence of prolonged openings (lasting several seconds) that were interrupted by few full rapid closings (Lingueglia et al 1995;Zhainazarov & Cottrell, 1998). Such prolonged openings were also a notable feature of many recordings from native neuronal FMRFamide-activated channels.…”

“…The observation that nLRFamide is an effective blocker, whereas FnLR is not, indicates that the carboxy, non-charged phenylalanine is important for providing appropriate spacial alignment of the charged guanidinium to cause blockade. The properties of the clone FaNaC and the native channel are very similar (Lingueglia et al 1995;Cottrell, 1997). The neuronal channel is thought to consist of only one type of subunit, FaNaC, four of which probably comprise the active channel (Coscoy et al 1998).…”

“…If each subunit possesses a single specific peptide-recognition site, each channel complex may have four such sites. On the other hand, concentration dependence studies of activation conform to a two-binding-site model: activation of the channel by FMRFamide appears to be co-operative, with a Hill coefficient of about 1·5 (Lingueglia et al 1995;Cottrell, 1997). Further, unitary current activity evoked by low and medium doses of FMRFamide and FLRFamide have been analysed satisfactorily assuming two peptide binding sites per functional channel (Zhainazarov & Cottrell, 1998), and a five-state model similar to that proposed for the nicotinic ACh receptor (see Colquhoun & Sakmann, 1985).…”

“…At that time it was unclear whether the two types of current represented distinct populations of ion channels, or a single type the properties of which became modified under certain conditions. The gene FaNaC has since been cloned from Helix brain by homology screening with primers based on a family of ion channels including the epithelial sodium channel (ENaC) and the degenerins of Caenorhabditis elegans, channels which were grouped based on their structure and their high sensitivity to block by amiloride (Lingueglia et al 1995). When mRNA transcribed from the clone (FaNaC) was expressed in Xenopus oocytes, FMRFamide activated Na¤-dependent, amiloride-sensitive, currents similar to those of the C2 neurone.…”