Abstract
VSOP is a protein that contains the voltage sensor domain but not pore domain (Sasaki et al, 2006). It exhibits most properties of native voltage-gated proton channels reported in phagocytes and microglia: voltage-dependent gating, proton-selective permeability and pH-regulated gating. Addressing questions how proton permeates and how voltage-dependent gating achieved in VSOP will lead to critical clues to understand general mechanisms of voltage sensor operation and ion permeation. For this purpose, it is important to define whether VSOP operates as monomer or oligomers. In this study, we expressed mouse VSOP in tsA201 cells and performed immunoblots. Antibody was raised against the C-terminal cytoplasmic region of mouse VSOP. Western blot analysis detected a major band, about 30 kDa, corresponding to monomer, whereas weak band corresponding to dimer (about 60 kDa) was also detected. When cysteine residue was introduced into the putative transmembrane region of S4, a key segment for voltage sensing, treatment with H2O2 induced upper band shift corresponding to crosslinked dimers. Such crosslinking still occurred when native cysteine was eliminated by point mutation and when the C-terminal cytoplasmic region was removed. Analysis by means of blue native PAGE also detected a band at a region higher than monomer. These suggest that VSOP operates as oligomer rather than monomer. Immunocoprecipitation experiments following cotransfection of myc-tagged VSOP and HA-tagged VSOP also supported this model. [J Physiol Sci. 2008;58 Suppl:S79]
