Sensing Charges of Ci-VSP

The Ciona intestinalis Voltage Sensor-containing Phosphatase (Ci-VSP) is a member of the Voltage Sensitive Phosphatase family that exhibits membrane potential-controlled enzymatic activity. Alignments of the amino acid sequence of Ci-VSP against Voltage Gated Channels (VGC) indicate that its Voltage Sensing Domain (VSD) is formed by four transmembrane segments. The putative fourth segment (S4) of Ci-VSP extends between the arginine (R) in position 217 and the glutamine (Q) in position 239, containing five arginines, which might be the voltage sensing charges. Although it has been shown that R229 and R232 are critical for voltage sensing in Ci-VSP (Murata et al., 2005), the role of the remaining charges is still unclear. To address this issue, we have performed a partial Histidine Scanning of the S4 of Ci-VSP, following the paradigm established for the VGC Shaker (Starace and Bezanilla, 2004). The voltage dependence of the sensing current of the R217H mutant was modulated by pH. Decreasing the external pH shifted the Q-V curve towards positive potentials, while a pH increase had the converse effect, consistent with the finding that neutralizing R217 (R217Q) produces a negative shift of the voltage dependence of Ci-VSP (Kohout et al., 2008). However, the total net charge of R217H did not change with pH, indicating that R217 does not participate in sensing the membrane potential. When the second arginine is replaced by histidine, the resultant mutant (R223H) exhibits a voltage dependent proton current which closes at positive potentials, resembling the current recorded from Shaker-W434F with its first gating charge replaced by histidine (R362H). This result strongly suggests that R223 has access to both the intra- and the extracellular media depending on voltage. Taken together, our results indicate that R223 is the most extracellularly located sensing charge of the Ci-VSP S4 segment. (Support: NIHGM030376

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing

A human phospholipid phosphatase activated by a transmembrane control module

In voltage-sensitive phosphatases (VSPs), a transmembrane voltage sensor domain (VSD) controls an intracellular phosphoinositide phosphatase domain, thereby enabling immediate initiation of intracellular signals by membrane depolarization. The existence of such a mechanism in mammals has remained elusive, despite the presence of VSP-homologous proteins in mammalian cells, in particular in sperm precursor cells. Here we demonstrate activation of a human VSP (hVSP1/TPIP) by an intramolecular switch. By engineering a chimeric hVSP1 with enhanced plasma membrane targeting containing the VSD of a prototypic invertebrate VSP, we show that hVSP1 is a phosphoinositide-5-phosphatase whose predominant substrate is PI(4,5)P(2). In the chimera, enzymatic activity is controlled by membrane potential via hVSP1\u27s endogenous phosphoinositide binding motif. These findings suggest that the endogenous VSD of hVSP1 is a control module that initiates signaling through the phosphatase domain and indicate a role for VSP-mediated phosphoinositide signaling in mammals

Stabilization of the relaxed state of the voltage sensing domain of Shaker

Segments S4 and S5 in Voltage Gated Channels potassium channels are contiguous and specific residues of these segments get in atomic proximity in a state-dependent way (Lainéet al., 2003; Lewis et al., 2008). In Shaker, the double mutation R362H+A419H stabilizes the conducting state of the channels when a metal bridge is formed in the presence of Zn2+ (Lainéet al., 2003). These results were obtained from ionic conduction experiments but gave no direct information on the dynamics of the Voltage Sensing Domain (VSD) of Shaker. As a proxy for the movement of the VSD, we studied the proton currents through the VSD that results by the double mutation R362H+A419H, on the ultra-fast-inactivating Shaker W434F. When the holding potential (HP) was 0 mV, the current-voltage relation of the proton current (Ip-V) was shifted towards negative potentials as compared to the Ip-V when HP was −90 mV, as expected from the relaxation that the VSD undergoes at maintained depolarization. When HP was 0 mV, the proton current was decreased and the Ip-V was further shifted by increasing the concentration of Ni2+ or Zn2+ (10 μM-100 μM). In contrast, no changes were observed in the Ip-V voltage dependence with Ni2+ or Zn2+ when holding at −90 mV. In the presence of Ni2+ or Zn2+ the proton current showed a second slower kinetic component, whose relative amplitude was increased with an increase in Ni2+or Zn2. Fluorescence recordings with a probe in M356C showed that Zn2+ decreased the rate of TMRM dequenching when pulsing to negative potentials from an HP of 0 mV, consistent with the proton current results. These observations indicate that the metal bridge between R362H and A419H stabilizes the relaxed state of the VSD (Support NIHGM030376)