Sequence alignment and function comparison of Ci-VSD with existing VSDs. (A) Gene of Ci-VSP consists of N-terminal, voltage sensing domain, a linker and phosphatase domain. The first 260 residues, indicated by the arrow on the top, is our current interest for biochemical preparation. Sequence of the fourth transmembrane segment (S4) of Ci-VSP was aligned with homologous sequences from voltage-gated ion channels. The positive charged residues (R/K) were distributed at every third position, and primarily responsible for voltage sensing and highly conserved. (B) Relationship between the charge movement and the test pulse amplitude (Q-V curve) of VSDs from a typical potassium channel Shaker (black) and Ci-VSP (red). The curves was simulated with V1/2 = −48 mV and z = 4.7 for Shaker (38), and V1/2 = 71.8 mV and z = 1.1 for Ci-VSP (3). The dotted line at 0 mV indicates the potential states of VSDs under biochemical conditions in absence of asymmetric voltage across the proteins: Shaker’s VSD at activated state (or Up state); Ci-VSD at resting state (or Down state).