FMRFamide-activated Ca2+ channels in Lymnaea heart cells are modulated by "SEEPLY," a neuropeptide encoded on the same gene

Abstract

The cell-attached, patch-clamp technique was used to investigate the modulatory role of the neuropeptide SEQPDVDDYLRDVVLQSEEPLY ("SEEPLY") on FMRFamide-activated Ca2+ channels in isolated Lymnaea heart ventricular cells. Both SEEPLY and FMRFamide are encoded on the same neuropeptide gene and are coexpressed in a pair of excitatory motor neurons that innervate the heart. FMRFamide applied alone was capable of significantly increasing the P(open) time of a Ca2+ channel in isolated heart muscle cells. However, SEEPLY applied alone did not significantly alter the basal level of Ca2+ channel activity in the same cells. Repeated applications of FMRFamide (15 s every min) resulted in a progressive reduction in the number of Ca2+ channel openings and the overall P(open) time of the channel. The fifth successive 15-s application of FMRFamide failed to cause the Ca2+ channels to open in the majority of cells tested. When FMRFamide and SEEPLY were repeatedly applied together (2-min applications every 4 min) the FMRFamide-activated Ca2+ channels continued to respond after the fifth application of the two peptides. Indeed channel activity was seen to continue after repeated 2-min applications of FMRFamide and SEEPLY for as long as the patch lasted (</=60 min). As well as preventing the loss of response to FMRFamide, SEEPLY was also capable of both up- and down-regulating the response of the Ca2+ channel to FMRFamide. The direction of the response depended on the P(open) time of the channel before the application of SEEPLY. When the P(open) time for the FMRFamide-activated channel was initially 0.004 +/- 0.002 (means +/- SE), subsequent perfusion with a mixture of FMRFamide and SEEPLY produced a statistically significant increase in Ca2+ channel activity (13 cells). In two cells where no channel activity was observed in response to an initial application of FMRFamide, superfusing the heart cells with a mixture of FMRFamide and SEEPLY induced openings of the Ca2+ channel. When the P(open) time of FMRFamide-induced Ca2+ channel openings was 0.058 +/- 0.017 the subsequent application of a mixture of SEEPLY and FMRFamide caused a statistically significant decrease in Ca2+ channel activity (8 cells). As up- and down-regulation of FMRFamide-activated Ca2+ channel openings by SEEPLY were observed in the same cells (8 cells), this suggested that corelease of the two peptides might act together to regulate the level of Ca2+ channel activity within a defined range.