FMRFamide-Activated Ca²⁺Channels inLymnaeaHeart 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.

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T-type Channels Become Highly Permeable to Sodium Ions Using an Alternative Extracellular Turret Region (S5-P) Outside the Selectivity Filter

“…Cut up ventricles were placed in 0.5 mM Ca 2ϩ Leibowitz medium containing gentamycin (400 mg/ml) and 30 mM glucose, trypsinized (0.25% (w/v) trypsin, Sigma T9201) for 10 min, and then treated with 0.1% collagenase (Sigma type II) in 0.5 mM Ca 2ϩ Leibowitz medium for 30 min. The digested hearts were then washed three times with 3.5 mM Ca 2ϩ Leibowitz medium and then plated on acidetched circular glass coverslips (16) and left for 24 h to adhere at room temperature in 3.5 mM Ca 2ϩ Leibowitz medium containing 400 mg/ml gentamycin, 30 mM glucose, and 2% fetal bovine serum, before patch clamp recording 24 h later.…”