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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

View larger version (36K): [in a new window]   Fig. 1. (A) Acetylcholine (nicotinic) receptor -subunit of Ascaris suum (ASAR-1 GenBank Accession No. AJ011382) and protein kinase phosphorylation sites. The -subunit combines with other subunits to form the pentameric receptor ion-channel membrane protein. The regions TM1, TM2, TM3 and TM4 are transmembrane regions. The region between TM3 and TM4 is exposed to the cytoplasm and contains consensus serine (S), threonine (T) and tyrosine (Y) phosphorylation sites. The structural motifs S, T or Y are consensus sites recognized by kinases: C, protein kinase C; A, protein kinase A; G, protein kinase G; T, tyrosine kinase; and CaM, Ca2+/calmodulin kinase. (B) Representative responses to levamisole (Lev) and acetylcholine (ACh). The amplitude of the first peak was measured for experimental purposes. The secondary component or slow-phase responses to levamisole and acetylcholine (arrows) were not always present but were seen in approximately one-third of the responses; they were more labile than the first component and may have arisen as a secondary effect of calcium entry following stimulation of the nicotinic receptors, or following stimulation of a metabotrophic (muscarinic) receptor (Hwang et al., 1999).

View larger version (30K): [in a new window]   Fig. 2. Effects of H-7. (A) Control levamisole (Lev) response and responses in the presence of 30 µmol l-1 H-7. Notice that there is a transient response following the application of H-7 and that the levamisole responses in the presence of H-7 are only a slightly lower amplitude than the control. (B) Effect of H-7 on the membrane potential response to 10 µmol l-1 acetylcholine (ACh). Representative recording, control acetylcholine response and responses in the presence of 10 µmol l-1 H-7 are shown. Notice that the acetylcholine responses in the presence of H-7 are the same size as the control responses.

View larger version (37K): [in a new window]   Fig. 3. Effects of staurosporine. (A) Two control levamisole (Lev) responses and two smaller responses in the presence of 1 µmoll-1 staurosporine. (B) Control acetylcholine (ACh; 10 µmoll-1) response and responses in the presence of 1 µmoll-1 staurosporine. Acetylcholine responses in the presence of staurosporine are smaller than the control responses.

View larger version (40K): [in a new window]   Fig. 4. Effects of KN-93, a selective Ca2+/calmodulin kinase II (CaM kinase II) antagonist. (A) Two control responses to 10 µmoll-1 levamisole (Lev) and two responses in the presence of 10 µmoll-1 KN-93. Notice that the peak responses in the presence of KN-93 are smaller than the control responses. (B) Effect of KN-93 on acetylcholine (ACh) is slowly reversible. The figure illustrates the antagonistic effect of 10 µmoll-1 KN-93 on the peak membrane potential responses to 10 µmoll-1 acetylcholine. Notice that 20 min was required in this preparation before the response to acetylcholine was clearly returning towards the control level. This may reflect the lipophilic nature of KN-93 and the long time required to wash the drug from the preparation.

View larger version (28K): [in a new window]   Fig. 5. Potentiating effect of Ca2+ on the levamisole concentration—response relationship. (A) Effect of different concentrations of levamisole applied in Ascaris peri-enteric fluid (APF) bathing solution (6 mmol l-1 Ca2+). (B) Effect of different concentrations of levamisole in low-Ca2+ APF (Ca2+ replaced with Mg2+). (C) Concentration—response plots of the effect of levamisole peak membrane potential responses in normal APF (Lev+Ca) and low-Ca APF (Lev—Ca). Best fit to response plot in the presence of Ca2+ in normal APF had an EC50 of 1.2 µmol l-1, a Hill slope of 1.21 and a maximum response of 7.1 mV. In low-Ca2+, the best-fit EC50 was 3.1 µmol l-1, the Hill slope was 3.9 and the maximum response was 6.8 mV. Note that the presence of Ca2+ is associated with an increase in the size of the response. The effect of Ca2+ on the response was statistically significant (P=0.045, analysis of variance).

View larger version (39K): [in a new window]   Fig. 6. Effects of genistein, a selective tyrosine kinase antagonist. (A) Two control responses to 10 µmol l-1 levamisole (Lev) and two responses in the presence of 90 µmol l-1 genistein. Notice that the responses in the presence of genistein are smaller than the control responses. (B) Two control responses to 10 µmol l-1 acetylcholine (ACh) and two responses in the presence of 90 µmol l-1 genistein. The effect is a small, statistically significant reduction in the size of the peak (see Table 1).

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