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 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.