Characterisation of activation and inhibition of the recombinant voltage-gated proton channel Hv1: functional reconstitution in unilamellare vesicles

The human voltage-gated proton channel (hHV1) regulates important physiological processes like the respiratory burst activity of white blood cells and the motility of spermatozoa by efflux of protons. Reconstitution of the recombinant full-length HV1 into small unilamellar vesicles provides a basis for studies of the electrophysiological properties and involved structural changes under well-defined conditions using a model membrane system. In the first section a protocol for the heterologous expression of HV1 fusion protein from Escherichia coli and solubilization from inclusion bodies by SDS has been established. Successful protein purification performed by metal ion affinity chromatography was confirmed by SDS-PAGE. Correct protein folding was verified by CD-spectroscopy and protein characterization was completed by western blotting and mass spectrometry. In the second section of this work the efficiency of detergent mediated reconstitution of HV1 in preformed unilamellar vesicles was analyzed by a pH-sensitive 9-amino-6-Chloro-2-methoxy (ACMA) fluorescence based proton flux assay. Effective reconstitution was accomplished by using cholesterol containing membranes reaching 76 ± 7 % of active proteoliposomes. Insertion of negative charged phospholipids allowed accelerating the proton translocation by accumulation of protons at the membrane surface. Furthermore the effect of temperature and of the potential channel inhibitor 2 guanidinebenzimidazole (2GBI) on the translocation rate were targeted. Q10-value of 1.99 and dissociation constant KD(2GBI) =88 ± 64 µM were determined and are in good agreement with the biophysical properties of native HV1 proton channels. Modifying the assay by use of lipid coupled pH-sensitive fluorophore Oregon Green 488-DHPE (OG488) in membranes (POPC/POPS/Chol/OG488-DHPE, 54:25:20:1) allowed to correlate fluorescence intensity and intravesicular pH-shift. The pH-shift was quantified to be around 0.11 pH units leading to a conductance of 0.06 fS. This is conform to in vitro studies of heterologous expressed Hv1.[1] The OG488-assay also allowed to estimate the KD(2GBI) = 47 ± 18 µM nearly as accurate as in vivo studies by Hong et al. do.[2] In the third section effective preparation of tethered bilayer lipid membrane (tBLM) by spreading of proteoliposomes onto a mixed monolayer of dihydrocholesteryl (2-(2-(2-ethoxy)ethoxy)ethanethiol and 6 mercaptohexanol was established. Formation of the lipid bilayer was confirmed via impedance and surface-enhanced infrared absorption (SEIRA) spectroscopy. While characterization of the electrophysiological properties of the HV1 failed due to the indistinguishability of HV1 and blank measurement SEIRA spectroscopy verified α-helical structure of the tBLM inserted HV1 channel. Thus providing the basis for further spectroelectrochemical studies in order to gain insight into the gating mechanism by drawing conclusion from voltage-dependent conformational changes of amid I band