Peptide potentiation of calcium channel activity can be seasonally variable.

Using the single-channel patch-clamp technique we have recently shown that the peptide cotransmitter proctolin can potentiate the activity of a large (38 pS in DVmmoir Ba; 26pS in m m m o l l " 1 Ca), depolarization-inducible Ca channel in the plasma membrane of the crayfish tonic flexor muscle (Bishop et al. 1991). Parallels between the modulatory effects of the peptide on channel activity and tension generated in the muscle suggest that this channel activation is probably partly responsible for the amplification of tension by proctolin (Bishop et al. 1991). Our initial studies of this phenomenon were performed during the spring and summer of 1988, using excised, inside-out patches. During that time, we found that the addition of 5xlO~molP proctolin to the bath at least 5min prior to seal formation (this dose gives a maximal response) caused a large increase in the proportion of patches with active channels and enhanced the single-channel open probability, Po, by as much as 40-fold. (Ca 2+ channels remained remarkably active in these excised patches: there was usually no obvious diminution in channel activity for the duration of the recordings; Bishop et al. 1991.) Identical results were obtained during the spring of 1989. In this communication we show that, during the winter months between these two periods, proctolin had virtually no effect on channel activity, indicating a seasonal variability in the potentiating effect of the peptide. Our studies were performed on 5-7.5 cm adult crayfish, Procambarus clarkii, supplied by Niles Biological, Sacramento, which obtained animals from local collectors. Animals were maintained in laboratory tanks (from several days up to 2 months) before use. Tonic flexor muscles were isolated (Fig. 1A) and treated with 0.05-0.15% collagenase IA for 20min. Muscles were bathed in DTmmoll" BaCl2 (to enhance Ca 2+ channel conductance), lmmoll" CaCl2, 2 .6mmoir 1 MgCl2 and lO.lmmoll" 1 Trizma (pH7.0). Patch pipets contained 205mmoll~ sodium gluconate, 2.6mmoll~ CaCl2, 2.6mmoll~ 1 MgCl2 and lO.lmmoll" Trizma (pH7.0). Experiments were performed at 21 °C, using the inside-out patch configuration. This configuration made it possible to identify Ca currents easily as inward currents in depolarized patches (see Bishop et al.