Figure 1.
Relationship between HVRP1 and other VSD-containing proteins.
A) Modular organization of HVRP1 compared to known voltage sensing proteins. The Shaker potassium channel was chosen as an example of protein containing both a VSD and a pore domain. C2D: C2 domain, CCD: coiled-coil domain, PhosD: phosphatase domain, PD: pore domain, TD: tetramerization domain. B) Phylogram showing amino acid sequence relationship between full-length VSP/TPTE/TPTE2 phosphatases, Hv1 channels, and HVRP1 proteins. See Methods for details. Ap: Anas platyrhynchos, Bt: Bos taurus, Ci: Ciona intestinalis, Dr: Danio rerio, Gg: Gallus gallus, Hs: Homo sapiens, Mb: Myotis brandtii, Mo: Metaseiulus occidentalis, Oa: Ornithorhynchus anatinus, Oo: Orcinus orca, Ps: Pelodiscus sinensis, Sp: Strongylocentrotus purpuratus, Tt: Tursiops truncates, Xt: Xenopus tropicalis. C) Hydrophobicity plot (generated with TopPred, Institute Pasteur, [53]), comparing human Hv1 and HVRP1 and showing relative positions of transmembrane helices S1–S4 (identified with SOSUI, Nagoya University, [54]). D) Sequence alignment of human Hv1 and HVRP1. Only regions containing segments S1 through S4 are shown.
Figure 2.
Tissue distribution of HVRP1 transcript.
A) HVRP1 expression in human tissues assessed by Affymetrix microarray analysis. HVRP1 tissue distribution was compared to the distributions of human Hv1 (HVCN1) and TPTE. Red and green signals indicate up-regulated and down-regulated expression, respectively. Struct.: skeletal muscle, adipose tissue, skin. CVS: heart and blood vessels. Resp.: respiratory system. Endo.: endocrine organs. Urinary & Repro.: urinary & reproductive systems (male and female). Immun.: immune tissues. Leuk.: peripheral white blood cells. Embr.: embryonic tissues (see also Table S1). B) Levels of HVRP1 transcript in total RNA extracts from the indicated human tissues measured by qRT-PCR. Levels are reported as relative expression units (RU) in relation to the housekeeping control gene beta-actin. Water was used as negative control. Error bars are S.E.M., n = 3. C) In situ hybridization on a 20-µm thick sagittal section of an adult mouse brain cerebellar region, using a 600-bp riboprobe targeting the HVRP1 mRNA. Positively stained regions are dark purple. Left panel: antisense probe. Right panel: control sense probe. Scale bars: 1 mm.
Figure 3.
Cellular and subcellular localization of the HVRP1 protein.
A) Immunohistochemical analysis of HVRP1 distribution in cerebellar tissue. Cerebellar cortical section from a wild-type rat stained with polyclonal antibody raised against HVRP1 (red). GC: granule cell layer, PC: Purkinje cell layer, ML: molecular layer, scale bar: 50 µm. B) Higher magnification of granule neurons expressing HVRP1, from (A). Scale bar is 10 µm. C) Ultrastructural localization of HVRP1 in the rat cerebellar GC layer visualized with the pre-embedding immunogold method. Positive labeling is in black, in the glomeruli, at the dendritic claws of granule cells surrounding mossy fiber terminals. Scale bar is 0.1 µm. D) Basic circuit diagram of the cerebellar cortex showing the location of HVRP1 in the dendrites and cell bodies of GCs. GO: Golgi cell, GR: glomerulus, MF: mossy fiber, PC: Purkinje cell. E) Confocal images of cultured mouse cerebellar granule neurons fixed and immunostained at 8 DIV. HVRP1 signal is shown in red (antibody dilution 1∶1000), DAPI signal is in blue. F) Staining as in (E) but with anti-HVRP1 antibody at dilution 1∶500 pre-incubated with HVRP1 antigen peptide (see Methods). Scale bars are 10 µm. G) Confocal image of live HEK293A cells expressing recombinant EGFP-tagged human HVRP1 (green) and labeled with the plasma membrane marker FM-464 (red). Scale bar is 10 µm. H) Western blot of total protein extracts from HEK293A cells transfected with hHVRP1 (H) and non transfected (N). AP indicates pre-incubation of anti-HVRP1 antibody with antigen peptide.
Figure 4.
Comparison between the VSD conducting properties of Hv1 and HVRP1.
A) Schematics of constructs used for the comparison (B–C). Black arrowheads indicate positions of the cysteine substitutions for fluorophore attachment (D). B) Proton currents from the indicated proteins expressed in a Xenopus oocyte measured in response to membrane depolarization by two-electrode voltage clamp. The test voltage was increased from 0 to +140 mV in 20 mV steps. Holding potential was −80 mV. Currents measured with HVRP1 are indistinguishable from the background. C) Quantification of test currents measured at +120 mV from traces like those shown in (B). Box indicates median ± S.D., whisker shows range. Individual measurements are shown as gray circles, n = 9–15. D) Fluorescence changes (expressed in arbitrary units) from TAMRA-MTS-labeled oocytes expressing the indicated proteins with or without cysteine substitution (shown in black and light gray, respectively). Substituted positions were: H193 in Hv1 and S196 in HVRP1. HVRP1V/D contained the additional mutation V111D. The membrane was depolarized to +120 mV from a potential of −100 mV. E) G–V relationship for the Hv1NCHVRP1 chimera compared to the G–V of Hv1 wild type and the Hv1NCCiVSP chimera. Error bars are S.E.M., n = 5. Normalized conductances were measured from tail currents recorded in inside-out patches from oocytes. Curved line for Hv1NCHVRP1 represents a Boltzmann fit with parameters: V1/2 = 40±3 mV, and slope = 12.8±1.1 mV. Curves for Hv1 and Hv1NCCiVSP represent previously published G-Vs with: V1/2 53±3 mV, slope 11.6±0.6 mV, and V1/2 68±2 mV, slope: 15.0±0.2 mV, respectively [16]. F) Example of deactivation kinetics of Hv1NCHVRP1 (teal) compared to Hv1 (red) and Hv1NCCiVSP (orange). Tail currents were measured in response to a voltage step from +140 mV to −80 mV and normalized to the maximal current.
Figure 5.
Consequences of increasing VSD similarity between Hv1 and HVRP1.
A) Sequence alignments of transmembrane segments S1 through S4 of human HVRP1 and Hv1 channels from three different species. Residues of the same category (see text) are shown in gray background. Category mismatches have white backgrounds. Mutated residues in HVRP1* are shown in red. B) Quantification of test currents measured at +120 mV in oocytes expressing the indicated proteins (conditions as in Fig. 4B–C). Hv1 and HVRP1 are positive and negative controls, respectively. Box indicates median ± S.D., whisker shows range. Individual measurements are shown as gray circles, n = 5–12. C) Confocal image of live HEK293A cells expressing recombinant EGFP-tagged HVRP1* (green) and labeled with the plasma membrane marker FM-464 (red). Scale bar is 10 µm.