Deuteration as a tool in investigating the role of protons in cell signaling

BackgroundThe mechanisms underlying the inhibitory effects of deuterium oxide (D2O; heavy water) are likely to provide insight into the fundamental significance of hydrogen bonds in biological functions. Previously, to begin elucidating the effect of D2O on physiological functions in living cells, we studied the effects of D2O on voltage-sensitive Ca2+ channels in AtT 20 cells and showed that actin distribution, Ca2+ currents, and β-endorphin release were affected. However, the molecular mechanisms underlying the inhibitory effects of D2O in whole animals and living cells remain obscure, especially in the effects of D2O on the cell signaling.MethodsWe investigated the molecular mechanisms underlying the inhibitory effects of D2O on the IP3-mediated Ca2+ signaling pathway using Ca2+ imaging and micro-calorimetric measurements in mGluR1-expressing CHO cells.ResultsDHPG-induced Ca2+ elevations were markedly reduced in D2O. Moreover, the Ca2+ elevations were completely suppressed in H2O after receptor activation with DHPG in D2O, recovering gradually in H2O medium. Without prior stimulation in D2O, however, DHPG-induced Ca2+ elevations in H2O were not affected. Micro-calorimetric measurements showed reduced total DHPG-evoked heat generation in D2O, while initial heat production and absorption associated with receptor activation were found to be larger. The reduction of DHPG-induced Ca2+ elevation and heat generation in D2O medium may be due to decreased amount of IP3 by the reduced hydrolysis of PIP2.General significanceProtein structure changes due to the replacement of hydrogen with deuterium will induce the inhibitory effects of D2O by reduction of the frequency of –OH bonds.

Research Highlights
► DHPG-evoked Ca2+ influx was reduced quickly when proton was changed to deuteron.
► Hysteresis effect on acute D/H exchange associated with receptor activation.
► Heat production after receptor stimulation was markedly attenuated in D2O.
► Amount of IP3 may be decreased in D2O by the reduced hydrolysis of PIP2.