The first peptide-gated ion channel.

Patch-clamp experiments on the C2 neurone of Helix aspersa have shown that the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRF amide) directly gates a Naf channel. The channel is amiloride-sensitive. Activation of this channel is responsible for the fast excitatory action of the peptide. Using primers based on amiloride-sensitive epithelial Naf channels, a complete cDNA sequence (FaNaCh) was cloned and sequenced from a Helix library, The sequence is predicted to have just two membrane-spanning regions and a large extracellular loop. When expressed in Xenopus laevis oocytes, the channel responded to FMRFamide. Taken together, these data provide the first evidence for a peptide-gated ion channel. Comparison of the properties of the expressed FaNaCh with the native neuronal channel show small differences in the sensitivities to some drugs and in channel conductance. It is not yet clear whether the native channel is a homo-oligomer or comprises other subunits. The peptide FKRFamide is an effective antagonist of FMRFamide on the expressed and neuronal channels. Nucleotide sequences encoding similar channel proteins occur in neurones of species as dissimilar as man and Caenorhabditis elegans, Some channels are thought to be associated with mechano-sensation, at least one is a proton-gated channel and others may also be ligand-gated channels.</p

Domain near TM1 influences agonist and antagonist responses of peptide-gated Na(+) channels.

A molecular biological approach was used to analyse the importance of different amino acids for ligand activation and for determining the action of amiloride on peptide- (Phe-Met-Arg-Phe-NH2, FMRFamide)-gated Na+ channels, members of the degenerin/epithelial Na channel (DEG/ENaC) family. Amiloride is a low-affinity blocker of most DEG/ENa channels, but has an unusual enhancing effect on responses of some of them. Chimeras were expressed in Xenopus oocytes and studied electrophysiologically. Differences in properties of channels from Helix aspersa and Helisoma trivolvis highlighted a sequence of 50 residues of the extracellular domain, near the first transmembrane segment (TM1), that affected sensitivity to FMRFamide, and whether amiloride blocked or enhanced the response to FMRFamide. Comparisons of chimeras prepared from H. aspersa and the extracellular domains of two other species, Aplysia californica and Lymnaea stagnalis and the preparation of further constructs, showed that amino acids 128-134 in the H. aspersa sequence are important in determining the predominant effect of amiloride and influencing the EC50 of FMRFamide. The results also showed that amino acids in this region are influenced by amino acids in other regions of the extracellular domain so as to affect not only the magnitude of responses, but also their time course and desensitisation.</p

The first peptide-gated ion channel.

Patch-clamp experiments on the C2 neurone of Helix aspersa have shown that the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRF amide) directly gates a Naf channel. The channel is amiloride-sensitive. Activation of this channel is responsible for the fast excitatory action of the peptide. Using primers based on amiloride-sensitive epithelial Naf channels, a complete cDNA sequence (FaNaCh) was cloned and sequenced from a Helix library, The sequence is predicted to have just two membrane-spanning regions and a large extracellular loop. When expressed in Xenopus laevis oocytes, the channel responded to FMRFamide. Taken together, these data provide the first evidence for a peptide-gated ion channel. Comparison of the properties of the expressed FaNaCh with the native neuronal channel show small differences in the sensitivities to some drugs and in channel conductance. It is not yet clear whether the native channel is a homo-oligomer or comprises other subunits. The peptide FKRFamide is an effective antagonist of FMRFamide on the expressed and neuronal channels. Nucleotide sequences encoding similar channel proteins occur in neurones of species as dissimilar as man and Caenorhabditis elegans, Some channels are thought to be associated with mechano-sensation, at least one is a proton-gated channel and others may also be ligand-gated channels.</p

Domain near TM1 influences agonist and antagonist responses of peptide-gated Na(+) channels.

A molecular biological approach was used to analyse the importance of different amino acids for ligand activation and for determining the action of amiloride on peptide- (Phe-Met-Arg-Phe-NH2, FMRFamide)-gated Na+ channels, members of the degenerin/epithelial Na channel (DEG/ENaC) family. Amiloride is a low-affinity blocker of most DEG/ENa channels, but has an unusual enhancing effect on responses of some of them. Chimeras were expressed in Xenopus oocytes and studied electrophysiologically. Differences in properties of channels from Helix aspersa and Helisoma trivolvis highlighted a sequence of 50 residues of the extracellular domain, near the first transmembrane segment (TM1), that affected sensitivity to FMRFamide, and whether amiloride blocked or enhanced the response to FMRFamide. Comparisons of chimeras prepared from H. aspersa and the extracellular domains of two other species, Aplysia californica and Lymnaea stagnalis and the preparation of further constructs, showed that amino acids 128-134 in the H. aspersa sequence are important in determining the predominant effect of amiloride and influencing the EC50 of FMRFamide. The results also showed that amino acids in this region are influenced by amino acids in other regions of the extracellular domain so as to affect not only the magnitude of responses, but also their time course and desensitisation.</p

Fast silencing reveals a lost role for reciprocal inhibition in locomotion

Summary Alternating contractions of antagonistic muscle groups during locomotion are generated by spinal “half-center” networks coupled in antiphase by reciprocal inhibition. It is widely thought that reciprocal inhibition only coordinates the activity of these muscles. We have devised two methods to rapidly and selectively silence neurons on just one side of Xenopus tadpole spinal cord and hindbrain, which generate swimming rhythms. Silencing activity on one side led to rapid cessation of activity on the other side. Analyses reveal that this resulted from the depression of reciprocal inhibition connecting the two sides. Although critical neurons in intact tadpoles are capable of pacemaker firing individually, an effect that could support motor rhythms without inhibition, the swimming network itself requires ∼23 min to regain rhythmic activity after blocking inhibition pharmacologically, implying some homeostatic changes. We conclude therefore that reciprocal inhibition is critical for the generation of normal locomotor rhythm.Publisher PDFPeer reviewe

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

1. In Helix neurones high doses of Phe-Met-Arg-Phe-NH2 (FMRFamide) often evoke biphasic inward whole-cell currents with brief application, and suppression of the current with prolonged application. With outside-out patches, a transient early suppression of the unitary current amplitude was seen following application of high doses of FMRFamide.2. Continuous application of a concentration of FMRFamide from 30 mu M to 1 mM resulted in a reduction in the amplitude of the unitary currents and an increase in open state noise. There was also an increase in the occurrence of smaller, 'subconductance' currents with the higher concentrations of FMRFamide. Similar effects were seen with BMRFamide on FaNaC expressed in oocytes. The FMRFamide analogues FLRFamide and WnLRFamide were more effective in evoking the lower conductance state. These effects of agonist at high concentrations were voltage dependent suggesting channel block.3. A similar effect was seen when one of the related peptides FKRFarnide, FM(D)RFamide, nLRFamide or N-AcFnLRFamide was co-applied with a low FMRFamide concentration. However, the non-amidated peptides FKRF, FLRF and nLRF and also WMDFamide did not have this effect.4. The inhibition of unitary currents induced by amiloride was qualitatively different from that produced by FMRFamide analogues with no obvious occurrence of subconductance levels. FMRFamide-gated channels were also blocked by guanidinium, but only at very high concentrations.5. The results strongly suggest a partial inhibition of current flow through the FMRFamide-gated channel by some part of the agonist or the related antagonist peptide molecules.</p

Modulation of ligand-gated dopamine channels in Helix neurones

Dopamine gates a fast excitatory response in Helix C2 neurones. Whole cell, and multiple unitary dopamine-gated currents showed variable decay rates and desensitization properties, suggesting the presence of more than one channel type. Manipulation of internal free [Ca2+] by various procedures (external zero Ca2+ or 1 mM Co2+, prolonged depolarization, A23187, or flufenamic acid), affected both the amplitude and decay time for the response, and also suggested the presence of separate fast and slowly decaying components. Responses were prolonged by intracellular fluoride a Iron specific phosphatase inhibitor, and attenuated and shortened by the protein kinase inhibitors H7 and staurosporine, and the calmodulin inhibitor W7. Phorbol ester potentiated and prolonged the response and this effect was reversibly antagonized by the specific protein kinase C inhibitor chelerythrine. Different dopamine-activated unitary currents were distinguished in outside-out patches by conductance (5, 8, 12 and 15pS), rate of recovery from desensitization, and pattern of openings. Discrimination of slow and fast components of the response was possible with apomorphine, ADTN, and caffeine. Paradoxically the dopamine antagonists chlorpromazine and spiperone, but not dopamine itself, stimulated sustained activity of 5pS unitary currents which did not desensitize in outside-out patches. Modulation of different channels underlying the fast dopamine response by protein kinase C, and possibly other mechanisms, provides a potent means of controlling excitatory dopaminergic synaptic transmission.</p