Estimating the effect of the deletions on the probability of channel opening.

<p><i>Aa</i>, Schematic of the P/-8 voltage protocol. <i>Ab</i>, Representative current at full scale, which is expanded in panel <i>Ac</i>. <i>B,C</i> Representative gating current traces recorded during depolarizing voltage steps from −100 to ∼ +50 mV (reversal potential): WT Ca_v3.1 (<i>Ba</i>); GD1–2 (<i>Bb</i>); GD3–5 (<i>Bc</i>); WT Ca_v3.3 (<i>Ca</i>); ID1–2 (<i>Cb</i>); and ID3–5 (<i>Cc</i>). Vertical scale bar is same size for all six traces (0.1 nA), while the horizontal scale bar is 1 ms in <i>B</i> and 2 ms in <i>C</i>. Data were acquired at 20 kHz, filtered at 10 kHz, and represent the average of 20 runs. G_max vs. Q_max for WT Ca_v3.1 and GD1–2 (<i>D</i>), or WT Ca_v3.3 and ID1–2 (<i>E</i>). The slope of the linear regression fit provides an estimate of <i>P_o,</i> and in both cases the slope of the line fitting the D1–2 mutants was 2-fold higher than for WT (Ca_v3.1, 0.26±0.03, n = 9; GD1–2, 0.55±0.06, n = 6, P<0.001; Ca_v3.3, 0.12±0.01, n = 9; and ID1–2, 0.26±0.02, n = 6, P<0.05). The difference between Ca_v3.1 and Ca_v3.3 is also statistically significant (P<0.001, one-way ANOVA followed by Tukey's multiple comparison test, Prism).</p

Electrophysiological properties of Ca_v3.1, Ca_v3.3, and their deletion mutants.

<p>The <i>G_max</i> and <i>V_0.5</i> of activation were determined from the <i>I-V</i> protocol, and therefore have the same number of cells (n) in each measurement. The <i>G/Q</i> ratio was calculated for each individual cell, and then averaged. Statistical significance is denoted with asterisks, where three asterisks indicates P<0.001, two for P<0.01, and one for P<0.05.</p

Location of deletions in the I–II loop.

<p>(<i>A</i>), Schematic representation of the I–II loop connecting repeat IS6 to repeat IIS1 in Ca_v3.1 and Ca_v3.3. Deleted regions are shown as open boxes. (<i>B</i>), Alignment of the I–II loop of human Ca_v3 channels. Arrows indicate where deletions begin and end. Dashes represent gaps in the alignment. Amino acids are color-coded by their physical properties as follows: red, positively-charged; green, negatively-charged; blue, polar; and yellow, hydrophobic.</p

Effect of deletions on channel surface expression as measured by electrophysiology and luminometry.

<p><i>A,B,</i> The <i>I-V</i> data from each cell was fit with a Boltzmann-Ohm equation to calculate <i>V_0.5</i>, <i>k</i>, <i>G_max</i>, and <i>E_rev</i>. The value of <i>E_rev</i> was then used to calculate the chord conductance at each test potential. These data were then normalized for cell capacitance. Only the increase in GD3–5 conductance (G_max) was statistically different than control Ca_v3.1. <i>C,</i> Luminometric quantification of the expression levels of HA-tagged Ca_v3.1 and Ca_v3.3 channel variants at the membrane (non-permeabilized). Average relative light units (RLU) for WT channels before normalization were: Ca_v3.1, 2,867,417±551,473; and Ca_v3.2 2,533,150±353,578, n = 6 for both). To reduce the error between experiments the data were normalized to the membrane expression for the respective WT channel. Total expression of HA-tagged channels measured after Triton X-100 permeabilization normalized to membrane expression. Ratio of surface/total expression identifies an increase in membrane expression for GD3–5, and decreases in GD1–2, ID1–2, and ID3–5. Statistically significant differences from control wild-type channels are indicated (**p<0.01; ***p<0.001).</p

Effect of Ca_v3.1 I–II loop deletions on the voltage dependence of channel activation.

<p><i>A, B,</i> Normalized current traces recorded during depolarizing voltage steps from −80 to +30 mV (holding potential, −100 mV, except for GD1–2 mutant, which due to shifted inactivation was −110 mV) in WT Ca_v3.1 (<i>Aa</i>), GD1–2 (<i>Ab</i>), GD3–5 (<i>Ac</i>), WT Ca_v3.3 (<i>Ba</i>), ID1–2 (<i>Bb</i>) and ID3–5 (<i>Bc</i>). Thick gray lines represent the current at −50 mV, demonstrating the negative shift in voltage dependence of activation observed in the deletion mutants. Currents were normalized to the maximum peak current in that cell. Time calibration bar scale applies to all three sets of traces in each case. Peak current-voltage plots for either Ca_v3.1 and its deletions (<i>C</i>) or Ca_v3.3 and its deletions (<i>D</i>). Peak currents were normalized to the cell size as estimated by capacitance. Normalized current-voltage plots for either Ca_v3.1 and its deletions (<i>E</i>) or Ca_v3.3 and its deletions (<i>F</i>). Same symbol definition as in panels <i>C</i> and <i>D</i>. Smooth curves in <i>C–F</i> represent fits to the average data using a Boltzmann–Ohm equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002976#pone.0002976-AriasOlgun1" target="_blank">[4]</a>.</p

Effect of D1–2 deletions on kinetics of Ca_v3.1 and Ca_v3.3 kinetics.

<p><i>A</i>, <i>B,</i> Normalized current traces for Ca_v3.1 (thick gray line), Ca_v3.3 (dashed line), and ID1–2. Currents were recorded during step depolarizations to −10 mV. The same current traces are shown in <i>A</i> and <i>B</i>, but at a different time scale. In <i>A</i> the time scale is expanded to illustrate how ID1–2 activates as fast as Ca_v3.1, while in <i>B</i> a longer time scale is shown to illustrate how ID1–2 inactivates at a similar rate as WT Ca_v3.3. <i>C,</i> Average activation kinetics estimated using a 2 exponential fit to the raw current traces obtained during the <i>I-V</i> protocol. Data represent mean±s.e.m , and N is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002976#pone-0002976-t001" target="_blank">Table 1</a>. <i>D,</i> Average inactivation kinetics. Same symbol definition as in panel <i>C</i>.</p

Estimating P_o of wild-type and Bdel1 channels.

<p>(Aa) Exemplar gating current at reversal potential (∼65 mV) for WT channels expressed with β2a. (Ab) Ionic current trace from the same cell recorded during a depolarizing step to +30 mV from a holding potential of −90 mV. Exemplar gating (Ba) and ionic (Bb) currents for the Bdel1 deletion mutant (also with β2a). Scale bars represent the same units as in panel A. (C) Plot of G_max versus Q_max where each symbol represents an individual cell. Solid line represents the fit to the data with linear regression. The slope, G/Q, is proportional to maximal channel open probability.</p

Deletions in the linker between AID and IS6 (Bdels) affect β regulation.

<p>Panels A–D show data obtained with Bdel1, panels E–H show data obtained with Bdel2, and panels I–L show data obtained with Bdel3. (A, E, I) Peak current-voltage relationships normalized to cell capacitance for Bdels expressed with β2a or β3. In the absence of a β, only Bdel1 produced detectable currents. (B, F,J) Normalized current traces recorded during depolarizing steps to +20 mV from a holding potential of −90 mV. Residual current after either 350 ms (C) or 25 ms (G, K) of depolarization divided by the maximum inward current, and plotted against test potential. (D, H, L) Comparison of the β2a and β3 effects on steady-state inactivation for Bdels estimated using 15 s prepulses to varying potentials. Dotted lines represent β3 effect on steady-state inactivation for WT channel.</p

Ca_vβ subunit regulation of α₁2.2.

<p>(A) Average peak currents normalized to cell capacitance. Smooth curves represent fits to the average data using a Boltzmann-Ohm equation. The symbols defined in (A) apply to all panels. (B) Activation represented by the normalized conductance (G/G_max). (C) Normalized current traces obtained during 350 ms step depolarization to +20 mV from a holding potential of −90 mV. WT+β3 currents are represented by a thick grey line. (D) The residual current at 350 ms of depolarizing pulse was divided by the maximum inward current and plotted against the test potential. (E) Representative current traces obtained during a test pulse to +40 mV after 15 s prepulses to varying potentials from a holding potential of −90 mV. Traces recorded after prepulses to −60 and −20 mV are highlighted to emphasize the β3 induced shift in steady-state inactivation. Scale bar represents 20 ms and 200 pA. (F) Effects of β2a and β3 on steady-state inactivation. The mean normalized amplitude of the currents is expressed as a function of membrane potential and fit with a Boltzmann equation (smooth curves). Data represent mean±SEM, in which the number of cells used to calculate the average is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003560#pone-0003560-t001" target="_blank">Table 1</a>.</p

Electrophysiological properties of Bdel1, Bdel2, and Bdel3 channels and their regulation by β2a and β3.

<p>Data shown are mean±SEM from the number of cells shown in parentheses. R values were determined from test pulses to +30 mV. Statistical significance for the β effects on Bdel1 were determined with ANOVA. Statistical significance between either Bdel2+β2a and Bdel2+β3, or Bdel3+β2a and Bdel3+β3 were determined by Student's <i>t</i>-test.</p>†<p>Currents from Bdel2 and Bdel3 were completely inactivated by 350 ms, so the R₂₅ is reported.</p>**<p>P<0.01.</p