Alcohols increase calmodulin affinity for Ca2+ and decrease target affinity for calmodulin

Abstract

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.

Ethanol Modulates BKCaChannels by Acting as an Adjuvant of Calcium

“…Ethanol increases Ca 2ϩ -binding to calmodulin (Ohashi et al, 2004). It is conceivable that an equivalent ethanol location in slo1 high-affinity binding sites could also increase their affinity for the metal and thus facilitate Ca 2ϩ -driven gating, as demonstrated by the changes in K C /K O ( Fig.…”

Deficiency in calcineurin activity in liver transplantation candidates with alcoholic cirrhosis or hepatocellular carcinoma

“…Ethanol can inhibit CNA by different mechanisms (24, 25). First, a direct CPA inhibition was reported in vitro by ethanol in aqueous medium enhancing Ca 2+ affinity to calmodulin and decreasing the calmodulin affinity for CNA (26, 27). An ethanol concentration of 1 g/L inhibits in vitro nearly 10% of CPA.…”

Mapping the sevoflurane‐binding sites of calmodulin

“…; Ohashi et al. ) and saturation transfer NMR experiments on halothane–CaM mixtures (Streiff et al. ).…”