Single-channel currents of a peptide-gated sodium channel expressed in Xenopus oocytes

1. Single-channel recordings were made from outside-out membrane patches of Xenopus oocytes injected with the cDNA clone FaNaCh, which encodes a peptide-gated Na+ channel from Helix aspersa.

2. The natural peptides FMRFamide and FLRFamide only activated unitary currents in oocytes injected with FaNaCh; the EC50 values were 1.8 and 11.7 mu M, respectively.

3. The slope conductance of the channel was 9.2 pS for both peptides.

4. With FMRFamide, the open probability (P-0) of the channel was 0.06 at 0.3 mu M and 0.76 at 30 mu M, whereas for FLRFamide the open probability increased from 0.04 at 1.8 mu M to 0.49 at 50 mu M. The Hill coefficient was greater than 1 for both peptides;

5. High concentrations of each peptide evoked very fast flickering between open and closed states which led to decreased unitary current amplitude.

6. At low doses, brief single openings and bursts of longer openings occurred. With higher doses, the occurrence of the brief openings declined and the number of longer openings increased; the duration of the longer openings was shorter with FLRFamide than with FMRFamide.

7. For each peptide, frequency distribution histograms of open events were best fitted by the sum of two exponential components, suggesting the existence of two open states of the channel. Closed events were fitted by the sum of three components, suggesting the existence of three closed states.

8. The data were analysed according to a five-state model in which the brief openings correspond to a single liganded open form of the channel and the longer openings to a doubly Liganded open form. According to this interpretation, the greater whole-cell response observed with FMRFamide than with FLRFamide results mostly from a slower closing rate constant for the longer (doubly liganded) channel openings.