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Results: 5

1.
F <span style="font-variant: small-caps" class="small-caps">ig.</span>  2.

F ig. 2. From: Evolution of the Voltage Sensor Domain of the Voltage-Sensitive Phosphoinositide Phosphatase VSP/TPTE Suggests a Role as a Proton Channel in Eutherian Mammals.

Properties of the currents generated by Hs-TPTE/TPTE2 domains. Whole-cell patch-clamp currents were recorded from HEK293 cells transfected with Dr-VSP or with Dr-VSP chimeras following transplant of the S3-loop-S4 domains (yellow and orange helices, fig. 1b) from Hs-TPTE (Dr-VSPHsTPTE:173-213) or from Hs-TPTE2 (Dr-VSPHsTPTE2:155-195). (a) Voltage protocol used to elicit currents. (bd) Currents recorded in HEK293 cells transfected with (b) Dr-VSP (black traces), (c) Dr-VSPHsTPTE:173-213 (red traces), and (d) Dr-VSPHsTPTE2:155-195 (blue traces). Transient sensing currents were recorded during depolarization and repolarization steps. Secondary currents were detected in Dr-VSPHsTPTE:173-213 and Dr-VSPHsTPTE2:155-195 transfectants but not in cells transfected with Dr-VSP. Time and current scales are shown. (e) Conductance–voltage relationship for secondary currents of Dr-VSP (black), Dr-VSPHsTPTE:173-213 (red), and Dr-VSPHsTPTE2:155-195 (blue) are shown. Data represents the mean (±standard error of the mean) of 8–16 independent experiments.

Keith A. Sutton, et al. Mol Biol Evol. 2012 September;29(9):2147-2155.
2.
F <span style="font-variant: small-caps" class="small-caps">ig.</span>  4.

F ig. 4. From: Evolution of the Voltage Sensor Domain of the Voltage-Sensitive Phosphoinositide Phosphatase VSP/TPTE Suggests a Role as a Proton Channel in Eutherian Mammals.

Proton currents are due to a single histidine in TPTE/TPTE2. (a) Sequence of the S4 segment of Dr-VSP is shown, with positions in which histidine is present in Hs-TPTE/TPTE2 indicated (asterisks). (b) Current traces for Dr-VSP point mutations. The voltage protocol used to elicit currents was identical to that of figure 2a. R171H point mutation of Dr-VSP (purple traces) produced proton currents, whereas T156H (green traces) and S174H (gray traces) mutations failed to conduct currents. (c) Conductance–voltage relationship for point mutations of Dr-VSP: T156H (green circles), R171H (purple circles), and S174H (gray circles). Conductances of Dr-VSP (black dashed lines, obscured by gray circles), Dr-VSPHsTPTE:173-213 (red line), and Dr-VSPHsTPTE2:155-195 (blue line) are shown for comparison (redrawn from fig. 2e). Data represent the mean (±standard error of the mean) of 7–12 independent experiments.

Keith A. Sutton, et al. Mol Biol Evol. 2012 September;29(9):2147-2155.
3.
F <span style="font-variant: small-caps" class="small-caps">IG</span> . 5.

F IG . 5. From: Evolution of the Voltage Sensor Domain of the Voltage-Sensitive Phosphoinositide Phosphatase VSP/TPTE Suggests a Role as a Proton Channel in Eutherian Mammals.

Role of histidine in the generation of voltage-sensitive proton currents. The proton current conducted by Dr-VSPHsTPTE:173-213 is abrogated by mutation of histidine H207 of Hs-TPTE, as shown here for the case of an H207Q mutation of Hs-TPTE:173-213. (a) Domain structure of Dr-VSP, Hs-TPTE, and chimeric constructs. Color code: zebra fish domains, red; zebra fish interdomain sequences, yellow; human domains, blue; and human interdomain sequences, gray. Residues corresponding to position 171 of Dr-VSP shown as: Dr-VSP, R171; Hs-TPTE, H207; Dr-VSPHsTPTE:173-213, H171; Dr-VSPHsTPTE:173-213(H207Q), H207Q mutation of human sequence. (b) HEK293 cells transfected with Dr-VSPHsTPTE:173-213 exhibit asymmetric sensing currents (as in fig. 2 in the main text) followed by a secondary current attributed to proton conductance (black traces). However, secondary currents were not observed in cells expressing Dr-VSPHsTPTE:173-213(H207Q) (blue traces). (c) Voltage–conductance relationship of currents in HEK293 cells transfected with Dr-VSPHsTPTE:173-213 (black) or with Dr-VSPHsTPTE:173-213(H207Q) (blue). Data represent the means (±standard error of the mean, error bars are obscured by the symbols) of five separate experiments.

Keith A. Sutton, et al. Mol Biol Evol. 2012 September;29(9):2147-2155.
4.
F <span style="font-variant: small-caps" class="small-caps">ig.</span>  3.

F ig. 3. From: Evolution of the Voltage Sensor Domain of the Voltage-Sensitive Phosphoinositide Phosphatase VSP/TPTE Suggests a Role as a Proton Channel in Eutherian Mammals.

Secondary currents are due to proton conductance. Whole-cell patch-clamp experiments were carried out on HEK293 cells transfected with Dr-VSPHsTPTE:173-213. (ac) Reversal potential was determined using identical intracellular and extracellular ionic conditions except for a 1 pH unit gradient (pH values shown in inset figure of patch-clamped cell). (a) Voltage protocol. (b) Currents recorded using intracellular and extracellular NaCl solutions (black traces) and with replacement of NaCl with impermeant NMDG+/sulfonate solutions (blue traces). (c) Current–voltage relationships for cells in NaCl (black line) and NMDG+/sulfonate (blue line) media, based on traces shown in b. Solid horizontal line indicates zero conductance. (df) The effects of pH gradient on secondary current amplitude. (d) Voltage protocol. (e) Current traces were recorded under symmetrical ionic conditions except for the indicated pH gradients (pH values shown in inset figure of patch-clamped cells). (f) Conductance–voltage relationship as a function of pH gradient (6.5 in/7.5 out, gray; 7.0 in/7.0 out, green; and 7.5 in/6.5 out, red) based on traces shown in e. Data (c, f) represent the mean (±standard error of the mean) of 7–12 independent experiments.

Keith A. Sutton, et al. Mol Biol Evol. 2012 September;29(9):2147-2155.
5.
F <span style="font-variant: small-caps" class="small-caps">ig.</span>  1.

F ig. 1. From: Evolution of the Voltage Sensor Domain of the Voltage-Sensitive Phosphoinositide Phosphatase VSP/TPTE Suggests a Role as a Proton Channel in Eutherian Mammals.

Domain structure of VSP/TPTE proteins. (a) Members of the VSP/TPTE family contain a voltage sensor consisting of four transmembrane segments (S1–S4), followed by a phosphoinositide phosphatase domain and a C2 region. Intracellular and extracellular regions are based on the orientation of Shaker Kv channels. The paddle motif is identified in structural studies and consists of the S3b and S4a portions of the voltage sensor. Domain swapping experiments used the S3-loop-S4 segment. (b) A homology model of the Hs-TPTE voltage sensor, constructed on the basis of the Shaker K+ channel, the Kv2.1 paddle-Kv1.2 chimera, and the NavAb voltage-gated Na+ channel, was used to identify transmembrane segments (S1, blue; S2, green; S3, yellow; and S4 orange). The three histidine residues present in and near the S4 segment of Hs-TPTE but not present in non-eutherian VSP are shown in stick representation (numbering based on Hs-TPTE, GI: 109689707). This figure and supplementary figure 2 (Supplementary Material online) were made with PyMOL (The PyMOL Molecular Graphics System, Version 1.4.1; Schrödinger, LLC). (c) Alignment of S4 segments shows that the positions of basic residues (shaded in yellow) in VSP/TPTE family members and in a Shaker Kv channel are conserved (numbered 1–7 based on Shaker sequence). Sequences include: Drosophila melanogaster Dm-Shaker Kv (GI: 288442), Ciona intestinalis Ci-VSP (GI: 66391023), Danio rerio Dr-VSP (GI: 193248592), Homo sapiens Hs-TPTE (GI: 109689707), and Hs-TPTE2 (GI: 213972591). The positions of three histidine residues present in human TPTE/TPTE2 are indicated (shaded in green, numbered 8–10). Asterisks indicate arginine residues of Dm-Shaker Kv that yield proton currents when mutated to histidine (Starace et al. 1997; Starace and Bezanilla 2001).

Keith A. Sutton, et al. Mol Biol Evol. 2012 September;29(9):2147-2155.

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