Interaction of divalent metal ions with the carboxyl-terminal domain of human voltage-gated proton channel Hv1.

Abstract

The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His(244) or His(266) residue. The binding of Zn(2+) to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co(2+), Ca(2+), Ni(2+), Mg(2+), and Mn(2+). The strength of Ca(2+), Co(2+), Mg(2+), Mn(2+) and Ni(2+) binding to the site close to His(244) is stronger than that of these divalent metal ions binding to the site close to His(266).