Efficacy of Ca_V3.2 inhibition by <i>Lavandula steochas</i> and <i>Rosmarinus officinalis</i> is dependent of the resting membrane potential.

<p>Inhibition and washout of Cav3.2 channels by 20 μg/ml <i>Lavandula stoechas</i>, or 20 μg/ml <i>Rosmarinus officinalis</i> was examined for HPs of -100 mV and -80 mV. Each bar represents the average of five to six similar experiments. Data represent the mean ± SEM.</p

<i>Lavandula stoechas</i>, <i>Lavandula angustifolia Miller</i> and Linalool effects on steady-state inactivation of Ca_V3.2 channels.

<p>To measure steady-state inactivation (at TP -30 mV), cells were voltage-clamped for 5 seconds at potentials between -110 and -40 mV (10 mV increments). (<b>A</b>) Representatives traces before and after treatment with 30 μg/ml <i>Lavandula steochas</i> essential oil (<i>LS</i> EO). (<b>B</b>) Steady-state inactivation before and after 30 μg/ml <i>Lavandula stoechas</i> essential oil (<i>LS</i> EO). (<b>C</b>) Steady-state inactivation before and after 50 μg/ml <i>Lavandula angustifolia Miller</i> essential oil (<i>LA</i> EO). (<b>D</b>) Steady-state inactivation before and after 100 μM Linalool (~15 μg/ml). Normalized steady-state inactivation curve in the presence of the compounds are represented by dotted curves in panels B, C and D. Data represents the mean ± SEM (n = 5–6).</p

Ca_V3.2 channel inhibition by <i>Rosmarinus officinalis</i> essential oil.

<p>Dose-response curves for <i>Rosmarinus officinalis</i> essential oil (<i>RO</i> EO) (<b>A-B</b>). and Rosmarinic acid (<b>C-D</b>). Representative current traces before and after application of serial concentrations of <i>RO</i> EO are superimposed, as shown on the left panels of each concentration-response curve. Each point represents the mean ± SEM. n = 6. The IC₅₀ for Rosmarinic acid (49.9 μM) corresponds to ~18 μg/ml.</p

Ca_V3.2 channel inhibition by Linalool at various frequency of stimulation.

<p>(<b>A-B</b>) Representative examples of Cav3.2 current inhibition by 100μM Linalool after 5 s (near half inhibition by linalool) and 90 s (near maximum inhibition by linalool) at 1 Hz (A) and 0.033 Hz (B). (<b>C</b>) Percentage of Linalool inhibition at three the frequencies tested, 1 Hz, 0.2 Hz and 0.033 Hz. (<b>D</b>) Time course of Linalool inhibition (Time for 50% inhibition) at the three frequencies tested, 1 Hz, 0.2 Hz and 0.033 Hz.</p

Inhibition of Ca_V3.2 channel by <i>Lavandula</i> essential oils and Linalool.

<p>Dose-response curves of the inhibitory effect of <i>Lavandula steochas</i> (<i>LS</i>) (<b>A</b>) <i>Lavandula angustifolia Miller</i> (<i>LA</i>) <i>(officinalis)</i> (<b>B</b>) and Linalool (<b>C</b>) on Ca_V3.2 current. Inhibition of Ca_V3.2 channel currents was obtained by serial increase in concentrations of <i>Lavandula sp</i>. extracts. The IC₅₀ for Linalool (84 μM) corresponds to ~12.6 μg/ml. Percentages of inhibition were averaged and plotted against compound concentrations (right panels; n = 7–8). Each point represents the mean ± SEM.</p

Modulation of Ca_V3.2 channels by medicinal plant methanolic extracts.

<p>Whole-cell patch clamp recordings of T-type calcium current were obtained on HEK-293T cells stably expressing recombinant human Ca_V3.2 channels. Currents were elicited by stepping from a holding potential (HP) of -80 mV to a test pulse (TP) of -30 mV applied every 10 seconds. Effect of the methanolic extracts (30 μg/ml) of the medicinal plants <i>Lavandula stoechas</i> (<b>A-B</b>), <i>Rosmarinus officinalis</i> (<b>C-D</b>), <i>Ricinus cummunis</i> (<b>E-F</b>), or <i>Citrullus colocynthis</i> (<b>G-H</b>) are illustrated with representative current traces collected before (open triangle) and during bath application (filled triangle) of the extracts (left panels). The corresponding time plots (right panels) illustrate the time-course of the inhibitory effect and washout of the extracts. Each extract panel is representative of 6 to 7 experiments.</p

<i>Rosmarinus officinalis</i> and Rosmarinic acid affect steady-state inactivation but not activation of Ca_V3.2 channels.

<p>(<b>A</b>) Representative <i>I-V</i> relationship of Ca_V3.2 channels before and after treatment with 50 μg/ml <i>Rosmarinus officinalis</i>. (<b>B</b>) Representative curve of the steady-state inactivation before and after 50 μg/ml <i>Rosmarinus officinalis</i>. (<b>C</b>) Representative <i>I-V</i> relationship of Ca_V3.2 channels before and after 50 μM (~18 μg/ml) Rosmarinic acid. (<b>D</b>) The steady-state inactivation before and after 50 μM Rosmarinic acid. Normalized <i>I-V</i> and steady state inactivation curves in the presence of <i>RO</i> and RA are represented by dotted curves in the four panels. Data represents the mean ± SEM (n = 6–7).</p

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

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 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