NS1643 enhances the activity of T1019PfsX38 and Q1070X hERG channels associated with long QT syndrome type 2

Figures

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  Fig. 1. Action potential clamp measurements of the effects of NS1643 on wild-type-, T1019PfsX38- and Q1070X-hERG. A - F, Average whole-cell currents from cells expressing different channel variants in the absence and presence of NS1643 [10 µM]. We applied a ventricular AP clamp waveform (red plot in A) and modified the obtained recordings using P/4 leak subtraction protocol. G-I, Summary data of the repolarization current integrals (Iintegral) measured as the area under the curve between 300 and 380 ms (APD(75−95)) of the AP duration. The treatment points in G - I were corrected for NS1643-induced inhibition. VC, clamp voltage; n = 4–6, paired Student’s t-test. Other details as in the Methods and Results sections and Figure S1

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  Fig. 2. Application of NS1643 to untransfected HEK293 cells. A, B, Representative currents from HEK293 cells in the absence and presence of NS1643 [10 µM]. The voltage clamp protocol is shown above A. C, Currents obtained by subtracting B from A. (D) Summary of normalised current integral values (A/A(max)) plotted against the test potential. n = 5 each, * p < 0.05, paired Student’s t-test

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  Fig. 3. The voltage dependence of activation of wild-type-, T1019PfsX38- and Q1070X-hERG in response to NS1643. A – F, Representative currents from HEK293 cells expressing wild-type-, T1019PfsX38- or Q1070X-hERG under control and NS1643 [10 µM] treatment conditions. The voltage clamp protocol is shown above A. G – I, Summary data of normalised peak tail currents in response to different test potentials. The baseline zero current was set at the end of the brief 40 ms depolarisation segment to −40 mV before test potentials. Peak currents were normalised by division over maximal peak current before adding the drug (Imax0). The continuous and dotted lines are best fits to the average data using the Boltzmann equation. J – L, Box plots of the half maximum voltage (Vmid) of activation under control and treatment conditions. n = 3–5, other details as in Fig. 1

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  Fig. 4. Example recording to assess changes in WT-hERG activation in response to NS1643. A and B, Representative currents from HEK293 cells expressing wild-type-hERG under control conditions and after the addition of NS1643 [10 µM]. The voltage-clamp protocol is shown above A

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  Fig. 5. hERG activation kinetic in response to NS1643. Summary data of normalized macroscopic hERG activation extent (I/I(max)) plotted against the duration of the activation voltage step (80, 40 or 0 mV) and measured as peak inward tail currents at −100 mV. The continuous and dotted lines are best fits to the average data using exponential equations. I(max) were the maximal tail current values after activation at 80 mV for 5.120 s in the same cell. The bar graphs to the right summarize values of the activation rate constants under control and NS1643 treatment conditions. The voltage clamp protocol follows the same shape as shown in Fig. 1. Wild-type (n = 7–12); T1019PfsX38 (n = 4–11); Q1070X (n = 4–6)

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  Fig. 6. hERG channel deactivation rates in response to NS1643. A, Expanded tail currents recorded from WT-hERG channels before and after the addition of NS1643. WT-hERG currents were activated for 2 s at 50 mV and the tail was observed at −40 mV (as in Fig. 3A-F). Red and green lines are best 1- and 2-component exponential fits to the data. B, Average data of the deactivation rate constants obtained by fitting the decaying tail currents. The percentages above the bars correspond to the normalized amplitudes of the fitted exponential components. n = 5 each