Mutua J et al. (2014), Functional diversity of voltage-sensing phospha...

XB-ART-49644

Physiol Rep 2014 Jul 16;27:. doi: 10.14814/phy2.12061.

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Functional diversity of voltage-sensing phosphatases in two urodele amphibians.

Mutua J , Jinno Y , Sakata S , Okochi Y , Ueno S , Tsutsui H , Kawai T , Iwao Y , Okamura Y .

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Voltage-sensing phosphatases (VSPs) share the molecular architecture of the voltage sensor domain (VSD) with voltage-gated ion channels and the phosphoinositide phosphatase region with the phosphatase and tensin homolog (PTEN), respectively. VSPs enzymatic activities are regulated by the motions of VSD upon depolarization. The physiological role of these proteins has remained elusive, and insights may be gained by investigating biological variations in different animal species. Urodele amphibians are vertebrates with potent activities of regeneration and also show diverse mechanisms of polyspermy prevention. We cloned cDNAs of VSPs from the testes of two urodeles; Hynobius nebulosus and Cynops pyrrhogaster, and compared their expression and voltage-dependent activation. Their molecular architecture is highly conserved in both Hynobius VSP (Hn-VSP) and Cynops VSP (Cp-VSP), including the positively-charged arginine residues in the S4 segment of the VSD and the enzymatic active site for substrate binding, yet the C-terminal C2 domain of Hn-VSP is significantly shorter than that of Cp-VSP and other VSP orthologs. RT-PCR analysis showed that gene expression pattern was distinct between two VSPs. The voltage sensor motions and voltage-dependent phosphatase activities were investigated electrophysiologically by expression in Xenopus oocytes. Both VSPs showed "sensing" currents, indicating that their voltage sensor domains are functional. The phosphatase activity of Cp-VSP was found to be voltage dependent, as shown by its ability to regulate the conductance of coexpressed GIRK2 channels, but Hn-VSP lacked such phosphatase activity due to the truncation of its C2 domain.

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Species referenced: Xenopus
Genes referenced: kcnj6 pten rpe tbx2 tns1

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	Figure 1. (A) Multiple amino acid alignments of newt (Cynops pyrrhogaster), salamander (Hynobius nebulosus), frog (Xenopus laevis), and chick (Gallus gallus) VSPs. The transmembrane segments (S1–S4) are surrounded by round squares. The conserved arginines in the S4 segment are shown in red. The conserved active site of the phosphatase domain is shown in green. The C2 domain is shown in blue. Note that a part of C2 domain is absent in salamander VSP. (B) Dendrogram of amino acid sequences of VSP orthologs. Bootstrap value is indicated.
	Figure 2. Measurement of Off “sensing” currents in newt VSP (Cp‐VSP) and its R153Q mutant. (A) A pulse protocol. Voltage was evoked ranging from −60 to +140 by 10‐mV increments. (B) Representative traces of an uninjected oocyte. (C, D) Representative traces of Off “sensing” currents of wild‐type (WT) Cp‐VSP and its R153Q mutant, respectively. Red trace indicates the current at +80 mV. Both On “sensing” and Off “sensing” currents were observed in R153Q mutants. (E) Qoff‐V curves from WT Cp‐VSP and its R153Q mutant. Red curves indicate fit by a Boltzmann equation. In WT data, plots were not saturated. The value of charges at 140 mV was used for standardization of Qoff‐V curves for WT. V1/2 of the R153Q mutant was 18.1 ± 1.5 mV, n = 4. Error bars are mean ± SD. Charge was standardized by the charge value of Qoff at repolarization from +140 mV step pulse. (F) Alignment of amino acid sequences of S4 segment among Cp‐VSP, Hn‐VSP, Xl‐VSP, and Gg‐VSP and the position of R153 is shown by asterisk.
	Figure 3. Off “sensing” currents in salamander VSP (Hn‐VSP) and its R153Q mutant. (A) A pulse protocol. Voltage was evoked ranging from −60 to +140 by 10‐mV increments. (B, C) Representative traces of Off “sensing” currents of wild‐type (WT) Hn‐VSP and its R153Q mutant, respectively. Red trace indicates the current at +80 mV. Both On “sensing” and Off “sensing” currents were observed in the R153Q mutant. (D) Qoff‐V curves from WT Hn‐VSP and its R153Q mutant. Red curves indicate plots fitted by Boltzmann equation. V1/2 of the R153Q mutant was 37.0 ± 2.6 mV, n = 4.
	Figure 4. Measurements of phosphatase activity of newt VSP (Cp‐VSP). (A) Schematic representation of experimental GIRK2 read out of depolarization‐dependent VSP activity. PtdIns(4,5)P2 depletion leads to the closure of the GIRK2 ion channel. (B) A pulse protocol for measuring GIRK2 currents on newt and salamander VSPs. Interval voltages were set to 50, 65, and 90 mV. (C) Depolarization‐dependent phosphatase activity of Cp‐VSP recorded at 50, 65, and 90 mV. (D) The amplitudes of the inward GIRK2 currents were normalized to the value of the first current trace and plotted against time. Data in the plot are mean ± SEM, n = 6, 50 mV (circles), 65 mV (squares), and 90 mV (triangles). (E) Bar graph for the normalized current at 3 sec. Error bars are SEM, P = 0.68, P = 0.04, **P = 0.02.
	Figure 5. Measurements of phosphatase activity of salamander VSP (Hn‐VSP). (A) Phosphatase activity of Hn‐VSP recorded at 50, 65, and 90 mV. (B) Normalized current amplitudes plotted against time. Data in the plot are mean ± SEM, n = 4, 50 mV (circles), 65 mV (squares), and 90 mV (triangles). (C) Bar graphs for current amplitudes at 3 sec. Error bars are SEM, P = 0.99, P = 0.94, **P = 0.95.
	Figure 6. C2‐domain truncated Ci‐VSP and Cp‐VSP mutants lack phosphatase activity. (A, B) Sensing currents recording of C2‐domain truncation mutant of Ci‐VSP and Cp‐VSP, respectively. (C, D) Phosphatase activity of truncated mutant of Ci‐VSP and Cp‐VSP, respectively, measured at 65 mV. (E) Qoff‐V plots for truncated Ci‐VSP (squares) and Cp‐VSP (circles), n = 6 for both. (F) Normalized current amplitudes plotted against time, n = 4 for both Ci‐VSP (squares) and Cp‐VSP (circles). Data in the plots are mean ± SD.
	Figure 7. RT‐PCR results of the gene expression pattern of Cp‐VSP and Hn‐VSP. (A) Cp‐VSP (upper panel). β‐actin for the positive control (lower panel). E, eye; K, kidney; S, spleen; I, intestine; G, gut; M, muscle; B, brain; H, heart; L, lung; O, ovary; T, testis. (B) Expression of Cp‐VSP in eye and retinal pigment epithelium (RPE), but not in the neural retina (+ or − indicates PCR reaction with or without reverse transcriptase) (C) Hn‐VSP (upper panel). β‐actin for the positive control (lower panel). E, eye; K, kidney; S, spleen; I, intestine; G, gut; M, muscle; B, brain; H, heart; L, lung; O, ovary; T, testis. Arrows indicate band size.
	Figure 8. Positive‐going membrane potential controls sperm‐egg fusion in Hynobius nebulosus but not in Cynops pyrrhogaster. (A) A graphical representation of percent polyspermy block. When the eggs were clamped at membrane potentials >20 mV Hn‐sperms (curve) were prevented from fusing with the egg, whereas Cp‐sperms (dash line) were not (from Iwao and Jaffe 1989). (B) Cartoon showing the hypothesis of molecular mechanisms of polyspermy block, based on the ability of sperm to sense change in the egg membrane potential (adapted from Iwao and Jaffe 1989). After the first sperm is successfully fused with egg, the second sperm is prevented from fusion with the egg through VSP activities. In the second sperm, VSP, which could potentially be localized in sperm membrane, senses depolarized egg cell membrane through transient cell fusion and its phosphatase activity downregulates phosphoinositides which may be required for progression of cell fusion event (Ratzan et al. 2011).

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