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

The cell-attached, patch-clamp technique was used to investigate the modula tory role of the neuropeptide SEQPDVDDYLRDVVLQSEEPLY ("SEEPLY") on FMRFamid e-activated Ca2+ channels in isolated Lymnaea heart ventricular cells. Both SEEPLY and FMRFamide are encoded on the same neuropeptide gene and are coe xpressed in a pair of excitatory motor neurons that innervate the heart, FM RFamide applied alone wets capable of significantly increasing the P-(open) time of a Ca2+ channel in isolated heart muscle cells. However, SEEPLY app lied alone did not significantly alter the basal level of Ca2+ channel acti vity in the same cells. Repented 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 1.5-s a pplication of FMRFamide failed to cause the Ca2+ channels to open in the ma jority of cells tested. When FMRFamide and SEEPLY were repeatedly applied t ogether (2-min applications every 4 min) the FMRFamide-activated Ca2+ chann els continued to respond after the fifth application of the two peptides. I ndeed channel activity was seen to continue after repeated 2-min applicatio ns of FMRFamide and SEEPLY for as long as the patch lasted (less than or eq ual to 60 min). As well as preventing the loss of response to FMRFamide, SE EPLY was also capable of both up- and down-regulating the response of the C a2+ 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-(ope n) 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 mix ture of FMRFamide and SEEPLY induced openings of the Ca2+ channel. When the P-(open) time of FMRFamide-induced Ca2+ channel openings was 0.058 +/- 0.0 17 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 SEE PLY were observed in the same cells (8 cells). this suggested that coreleas e of the two peptides might act together to regulate the level of Ca2+ chan nel activity within a defined range.