Levamisole receptor phosphorylation: effects of kinase antagonists on membrane potential responses in Ascaris suum suggest that CaM kinase and tyrosine kinase regulate sensitivity to levamisole

Abstract

SUMMARY A two-micropipette current-clamp technique was used to record electrophysiological responses from the somatic muscle of Ascaris suum. Levamisole and acetylcholine were applied to the bag region of the muscle using a microperfusion system. Depolarizations produced by 10 s applications of 10 μmol l-1 levamisole or 20 s applications of 10 μmol l-1 acetylcholine were recorded. The effect on the peak membrane potential change of the kinase antagonists H-7, staurosporine, KN-93 and genistein was observed. H-7 (30 μmol l-1), a non-selective antagonist of protein kinases A, C and G but which has little effect on Ca2+/calmodulin-dependent kinase II (CaM kinase II), did not produce a significant effect on the peak response to levamisole or acetylcholine. Staurosporine (1 μmol l-1), a non-selective kinase antagonist that has effects on protein kinases A, C and G, CaM kinase and tyrosine kinase, reduced the mean peak membrane potential response to levamisole from 6.8 mV to 3.9 mV (P<0.0001) and the mean response to acetylcholine from 5.5 mV to 2.8 mV (P=0.0016). The difference between the effects of H-7 and staurosporine suggested the involvement of CaM kinase II and/or tyrosine kinase. KN-93, a selective CaM kinase II antagonist,reduced the mean peak response to levamisole from 6.2 mV to 2.7 mV(P=0.035) and the mean peak response of acetylcholine from 4.7 mV to 2.0 mV (P=0.0004). The effects indicated the involvement of CaM kinase II in the phosphorylation of levamisole and acetylcholine receptors. The effect of extracellular Ca2+ on the response to levamisole was assessed by comparing responses to levamisole in normal and in low-Ca2+ bathing solutions. The response to levamisole was greater in the presence of Ca2+, an effect that may be explained by stimulation of CaM kinase II. Genistein (90 μmol l-1), a selective tyrosine kinase antagonist, reduced peak membrane potential responses to levamisole from a mean of 6.4 mV to 3.3 mV (P=0.001). This effect indicated the involvement of tyrosine kinase in maintaining the receptor.