Abstract
The voltage-gated proton channel Hv1 has a voltage sensor domain but lacks a pore domain. Although the C-terminal domain of Hv1 is known to be responsible for dimeric architecture of the channel, its role and structure are not known. We report that the full-length Hv1 is mainly localized in intracellular compartment membranes rather than the plasma membrane. Truncation of either the N or C terminus alone or both together revealed that the N-terminal deletion did not alter localization, but deletion of the C terminus either alone or together with the N terminus resulted in expression throughout the cell. These results indicate that the C terminus is essential for Hv1 localization but not the N terminus. In the 2.0 A structure of the C-terminal domain, the two monomers form a dimer via a parallel alpha-helical coiled-coil, in which one chloride ion binds with the Neta atom of Arg(264). A pH-dependent structural change of the protein has been observed, but it remains a dimer irrespective of pH value.
Highlights
Voltage-gated proton channel (Hv channel) currents were observed first in snail neurons [1] and later found in many mammalian cells, such as alveolar epithelium cells of the lung, microglia of the brain, skeletal muscle, and many blood cells, including macrophages, neutrophils, and eosinophils [2,3,4,5]
Truncation of either the N or C terminus alone or both together revealed that the N-terminal deletion did not alter localization, but deletion of the C terminus either alone or together with the N terminus resulted in expression throughout the cell. These results indicate that the C terminus is essential for Hv1 localization but not the N terminus
Hv1 Is Mainly Located in Intracellular Sites, and the C Terminus Is Essential for Its Localization—HeLa cells grown on glass coverslips were transiently transfected with the protein expression plasmids as described under “Experimental Procedures,” respectively, and observed by enhanced green fluorescent protein (EGFP) fluorescence under DM RXA2 microscopy
Summary
Expression Vectors—Two different kinds of Hv1 expression plasmids were constructed. The Hv1 cDNAs that were full-. Fluorescence of EGFP and Rhodamine Phalloidin—HeLa cells on glass coverslips were transfected with the protein expression plasmids for 48 h, washed with serum-free Dulbecco’s modified Eagle’s medium, and subsequently incubated with rhodamine phalloidin (Invitrogen) that binds to F-actin to delineate the cell’s edges, in serum-free Dulbecco’s modified Eagle’s medium at 37 °C for 30 min, following the manufacturer’s protocol. Heavy atom derivative data were collected from a crystal soaked in cryosolution containing 1 mM HgCl2 for 12 h. The purified C terminus was dialyzed overnight against various pH value buffers containing 0.2 M NaCl and 0.5 mM dithioerythritol. Analytical Ultracentrifugation— The purified C terminus was dialyzed overnight against various pH value buffers containing 0.2 M NaCl and 0.5 mM dithioerythritol, as described above. The sedimentation equilibrium data were analyzed by using the XL-A data analysis software (Beckman, version 4.01)
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