Alcohols increase calmodulin affinity for Ca2+ and decrease target affinity for calmodulin.
It has been proposed that alcohols and anesthetics selectively inhibit proteins containing easily disrupted motifs, e.g., alpha-helices. In this study, the calcineurin/calmodulin/Ca(2+) enzyme system was used to examine the effects of alcohols on calmodulin, a protein with a predominantly alpha-helical structure. Calcineurin phosphatase activity and Ca(2+) binding were monitored as indicators of calmodulin function. Alcohols inhibited enzyme activity in a concentration-dependent manner, with two-, four- and five-carbon n-alcohols exhibiting similar leftward shifts in the inhibition curves for calmodulin-dependent and -independent activities; the former was slightly more sensitive than the latter. Ca(2+) binding was measured by flow dialysis as a direct measure of calmodulin function, whereas, with the addition of a binding domain peptide, measured calmodulin-target interactions. Ethanol increased the affinity of calmodulin for Ca(2+) in the presence and absence of the peptide, indicating that ethanol stabilizes the Ca(2+) bound form of calmodulin. An increase in Ca(2+) affinity was detected in a calmodulin binding assay, but the affinity of calmodulin for calcineurin decreased at saturating Ca(2+). These data demonstrate that although specific regions within proteins may be more sensitive to alcohols and anesthetics, the presence of alpha-helices is unlikely to be a reliable indicator of alcohol or anesthetic potency.
Ohashi
Ichiro
I
Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama, Japan.
Pohoreki
Roman
R
Morita
Kiyoshi
K
Stemmer
Paul M
PM
D000438:Alcohols / Q000737:chemistry / Q000378:metabolism*; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005454:Fluorescence Polarization; D011485:Protein Binding; D017433:Protein Structure, Secondary
D000438:Alcohols; D002147:Calmodulin; D019703:Calcineurin; D002118:Calcium