This study focuses on sequence changes that occur in and near the S4 segment of VSP/TPTE phosphatases during the evolutionary history of these proteins. The positions of positively charged residues in the S4 segment that account for voltage sensor function are conserved to a great extent within the VSP/TPTE family as well as between VSP/TPTE and corresponding segments of K+
channels and other cation channels (Worby and Dixon 2005
; Okamura 2007
). Yet, we noted sequence differences between human (Hs-TPTE and Hs-TPTE2) and zebra fish (Dr-VSP) members of the VSP/TPTE family, particularly regarding the introduction of one histidine residue in the S4 segment of human orthologs and two others that lie just outside S4. One of these histidines (Histidine-207 of Hs-TPTE; shown as H9 in ), when present in the context of Dr-VSP, converted that zebra fish protein into a voltage-gated proton current.
The VSP/TPTE family is conserved from ascidians to mammals. However, inspection of available genomic sequences revealed that histidine is not present at the position essential for proton currents in either nonmammals or in a prototherian mammal but instead is first observed during the radiation of eutherian mammals (sequences in ). It is retained at this position in eutherians from several superorders, suggesting that this may represent an ancestral form. Furthermore, gene duplication events in primates, including human, have resulted in paralogs that, in one case (TPTE2), remain an active phosphatase while, in another (TPTE), have lost catalytic activity due to sequence changes in the enzyme active site (Walker et al. 2001
; Leslie et al. 2007
). Yet both human paralogs retain histidine at the essential position in the S4 segment of the voltage sensor and both produce voltage-dependent proton currents in the experimental context studied here. These observations suggest that a selection pressure fixed histidine in this position. However, computational analysis failed to reveal positive selection of this residue (see Materials and Methods: Evolutionary Analysis), suggesting that this is a case of functional adaptation in the absence of sequence-based signals, as has been reported in other systems (Hughes 2008
; Yokoyama et al. 2008
). The physiological basis of this selection pressure has not been identified but may relate to the requirement for proton currents.
Sequences of S4 Segment of VSP/TPTEa
There are two models for the production of proton currents by voltage sensor domains. First is the case of HVCN1, which contains an S1–S4 voltage sensor domain but lacks an identified target domain such as the ion pore of cation channels or the catalytic domain of VSP/TPTE phosphatases (Ramsey et al. 2006
; Sasaki et al. 2006
). This channel accounts for the endogenous voltage-sensitive proton currents that have been detected in a wide range of eukaryotic cells (Capasso et al. 2011
). Proton currents are conducted along an immobilized water wire that penetrates through the sensor domain of HVCN1 but that is absent from Kv channels (Ramsey et al. 2010
; Wood et al. 2011
). Histidine residues are not present in the S4 segment of HVCN1. In addition, the position corresponding to the histidine that is required for proton currents through chimeric Dr-VSP does not play an essential role in HVCN1 channel activity (Ramsey et al. 2006
). Thus, it is likely that HVCN1 and chimeric Dr-VSP use different mechanisms to conduct protons.
Alternatively, proton currents can be generated through the voltage sensor domain of Drosophila
Shaker Kv channels following experimental replacement of certain conserved basic residues in the S4 segment with histidine (shown as asterisks, ). In those mutagenesis experiments, histidine was used to probe voltage-driven conformational changes. The proton currents that resulted are thought to reflect the movement of the introduced histidine from contact with the aqueous environment into the membrane electrical field during voltage-driven sensor activation, and once there to allow this titratable residue to function as a proton shuttle (Starace et al. 1997
; Starace and Bezanilla 2001
). Chemical probe studies suggest that the residue in Shaker Kv channels (lysine-380, shown as K-7 in ) that corresponds to the Hs-TPTE/TPTE2 histidine required for proton currents (histidine-207 in Hs-TPTE, shown as H-9 in ) may also move from contact with the aqueous environment into the focused electrical field during activation of the sensor domain (Elinder et al. 2001
). If similar movement also occurs in the voltage sensor of eutherian TPTE/TPTE2, where histidine has been fixed by evolution, then it could permit that residue to conduct protons across the field. Testing this suggestion will require the development of heterologous expression systems that traffic full-length Hs-TPTE/TPTE2 to the cell surface.
Our results suggest that proton currents are conducted by the voltage sensors of Hs-TPTE/TPTE2 and of many eutherian orthologs. However, this current requires strongly positive membrane potentials for activation; it is first observed at > +20 mV and full activation requires > +100 mV () in the heterologous expression system used here. Mammalian TPTE has been localized in the Golgi complex in spermatogenic cells (Guipponi et al. 2001
; Tapparel et al. 2003
) and in cell lines following transfection (Guipponi et al. 2001
; Walker et al. 2001
; Wu, Dowbenko, et al. 2001
). But the membrane potential of the Golgi complex is ~0 mV (Schapiro and Grinstein 2000
; Wu, Grabe, et al. 2001
; Maeda et al. 2008
) and may not be sufficient to activate the TPTE/TPTE2 voltage sensor. Similarly, Ci-VSP, which has been reported to be a flagellar plasma membrane protein of C. intestinalis
sperm (Murata et al. 2005
), activates at membrane potentials >0 mV (Sakata et al. 2011
), whereas the plasma membrane potential in the sperm of that ascidian is ~−50 mV and may hyperpolarize to ~−100 mV in response to factors released from eggs (Izumi et al. 1999
). Finally, X. laevis
VSP1 and Xenopus tropicalis
VSP activate at ~0 mV following heterologous expression, but the membrane potential of those sperm has not been reported (Ratzan et al. 2011
). In general then, VSP/TPTE orthologs have functional voltage sensors that have been shown in some cases to regulate catalytic activity (Murata et al. 2005
; Hossain et al. 2008
; Ratzan et al. 2011
), but it is uncertain whether the membrane potential conditions required for phosphatase activation occur under physiological conditions.
In summary, we report that a histidine residue is introduced into the cytoplasmic end of the S4 segment of the TPTE voltage sensor during an early stage of the eutherian radiation. This sequence change is fixed in TPTE and paralogs in most eutherian mammals. The presence of histidine at this position permits the voltage sensor domain to conduct voltage-sensitive proton currents.