Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Exemplar traces for a −30 mV outward tail protocol for the N-truncated wild-type jShak1, S2 single mutants and the S4 insertion mutants. (A) N-truncated wild-type jShak1 channels exhibit measurable outward tail currents during a −30 mV tail-step. For illustrative purposes, this simplified tail-current protocol (inset) evokes currents from a −90 mV holding potential to a range of 50 ms step depolarisations −90 to +90 mV in 10 mV steps followed by a 20 ms tail-step to −30 mV, and a return to −90 mV for 200 ms before the next sweep. Note: for energetic analysis, the protocol range was extended to a range of −140 to +90 mV for 50 ms using 2 mV steps to increase the detection of low probability opening required for energetic analysis. (B) Traces obtained for the N227E and N227D S2 mutants obtained as in A. (C) Traces obtained for the Arg-Ile-Phe (RIF), Gln-Ile-Phe (QIF) and Ile-Phe-Arg (IFR) S4 insertion mutants obtained as in A.

• Supplemental Figure S2 -

  Fig. S2. Example of data analysis used to determine gating charge. (A) A single exponential function inadequately fits the tail currents, giving a good fit at long times after the voltage shift but consistently falling below the data at short times after the voltage shift. (B) The sum of two exponentials, a kinetic result observed in many K_V1 channels, successfully fit the tail currents of jShak1 channels. (C) The parameters from the fit of the sum of two exponentials were used to calculate tail current amplitude at 2 mV intervals. Data points were fit to a Boltzmann curve and transformed to yield G/G_max as described in the Materials and methods section. (D) Graph of log (G/G_max) vs V for a Gln-Ile-Phe (QIF) mutant channel with multiple experiments pooled to maximize signal precision at low depolarization potentials. The limiting slope is used to determine the gating charge, avoiding the artifact of low values obtained from direct calculation of the charge from the Boltzmann slope factor that is caused by the multistate kinetic scheme of true voltage response of voltage-gated channels. (E) Expanded view of the log(G/G_max) vs V curve from A illustrating several of the fitted lines calculated using the sliding window analysis. (F) Graph of the slope calculated from the sliding 15-point window over the log(G/G_max) vs V curve shown in D as a function of position along the curve. This illustrates that the curve approaches a limiting slope at voltages with low total conductance, and illustrates the estimate of that asymptote as an average of the slope values within the near-asymptotic portion of the graph (red horizontal line labelled with the estimate of z_lim=3.27.

• Supplemental Figure S3 -

  Fig. S3. Limiting slope evaluation of gating charge for channels. Examples of the analysis used to estimate the gating charge for each of the 12 channels used in this study. Sliding window analysis was performed on log(G/G_max) vs voltage graphs for each of the recordings, as shown in Fig. S2. An example of one of the analyses for each of the channels is shown. The values of the slopes calculated from each of the sliding windows were then graphed against the midpoint of the voltage range for that window. As V becomes more negative the values of the slope asymptotically approach the values for z_lim. The red dashed lines indicate the average of the values of the points that make up the plateau. In all 12 cases these graphs reached a plateau.

• Supplemental Figure S4 -

  Fig. S4. Homology model of jShak1 based on the structure of RatK_V1.2. (A) Full alpha subunit complete with T1 domain and modelled loops RatK_V1.2 (orange) and jShak1 (blue). The placement of residues in the S1 and S3 helices was determined by comparing PredictProtein secondary structure predictions on K_V1.2 to the corresponding structural motifs in the crystal structure. The PredictProtein method pinpoints the location transmembrane helices to approximately 80% accuracy. The interhelical linkers were modelled purely from loop databases where the conformation selected gave the lowest energy tertiary structure for a given sequence that matched the secondary structure predictions for that region. The modelling of these structures was informed with information from electrophysiological and perturbation studies on a number of channels. (B) A larger view of A with the T1 domain removed.

• Supplemental Table S1 - Adobe PDF