Alcohols increase calmodulin affinity for Ca 2+ and decrease target affinity for calmodulin

Abstract

It has been proposed that alcohols and anesthetics selectively inhibit proteins containing easily disrupted motifs, e.g., α-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 α-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 α-helices is unlikely to be a reliable indicator of alcohol or anesthetic potency.