Sensing charges of Ci-VSP


Carlos A. Villalba-Galea: 0000-0002-6489-4651

Document Type


Conference Title/Conference Publication

Biophysical Journal


Biophysical Society 54th Annual Meeting


San Francisco, CA

Conference Dates

February 20-24, 2010

Date of Presentation







3, Supplement 1



First Page



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)