Functional Reconstitution of Purified Human Hv1 H+ Channels

Seok-Yong Lee, James A. Letts, Roderick MacKinnon
2009 Journal of Molecular Biology  
Voltage-dependent H + (Hv) channels mediate proton conduction into and out of cells under the control of membrane voltage. Hv channels are unusual compared to voltage-dependent K + , Na + and Ca 2+ channels in that Hv channel genes encode a voltage sensor domain (VSD) without a pore domain. The H + currents observed when Hv channels are expressed heterologously suggest that the VSD itself provides the pathway for proton conduction. In order to exclude the possibility that the Hv channel VSD
more » ... mbles with an as yet unknown protein in the cell membrane as a requirement for H + conduction we have purified Hv channels to homogeneity and reconstituted them into synthetic lipid liposomes. The Hv channel VSD by itself supports H + flux. Keywords Voltage-sensor; Membrane protein; Proton channel Voltage-dependent H + (Hv) channels were first described in snail neurons and have been characterized most thoroughly in mammalian cells by DeCoursey 1; 2 . Hv channels are involved in many cellular functions including acid extrusion and charge compensation during the phagocyte respiratory burst 3 . The recent discovery of Hv channel genes demonstrated that they encode membrane proteins that are related in amino acid sequence to voltage sensor domains (VSD) of voltage-dependent cation channels 4; 5 . Voltage-dependent cation channels contain a single central pore surrounded by four VSDs, which regulate opening and closing of the pore 6; 7 . It was therefore interesting to observe that Hv channels appear to consist of a regulatory (voltage-sensor) domain without a pore, and yet, when expressed in mammalian cells, they produce voltage-dependent H + currents with pharmacological properties expected of native Hv channels 4; 5 . Functional expression of a gene in cells leaves open the possibility that an unknown component provided by the cell is necessary to support function. Stimulated by our interest in voltage sensor proteins our laboratory has been studying Hv channels using biochemical methods with the ultimate goal to determine the atomic structure. Our expression and purification studies enable us to test the functional competence of the Hv VSD-like protein in a completely defined reconstituted state 8 .
doi:10.1016/j.jmb.2009.02.034 pmid:19233200 pmcid:PMC2778278 fatcat:j3zh66o35rgormx7eo47cfdbzq