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The Journal of Experimental Biology 205, 3979-3988 (2002)
Copyright © 2002 The Company of Biologists Limited

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

Sasa M. Trailovic, Alan P. Robertson, Cheryl L. Clark and Richard J. Martin^*

Department of Biomedical Sciences, Iowa State University, Ames, Iowa, IA 50011, USA

^* Author for correspondence (e-mail: rjmartin{at}iastate.edu)

Accepted 16 September 2002

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.

Key words: levamisole, kinase antagonist, H-7, KN-93, genistein, staurosporine, Ascaris summ, nematode, CaM kinase, tyrosine kinase, membrane potential response

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