Figure 4.
Nav1.3 (SCN3A) is the major contributor to inward Na+ current in mouse CCs.(A) Each point represents quantitative RT-PCR reactions run in triplicate for a single AM for the indicated Nav channel subunits from mouse AM, with message levels normalized to β-actin. (B) Similar reactions were run on rat AM. In both A and B, at least three AMs were examined for each message species. In A, P values (from ANOVA with Tukey’s post hoc test) <0.05 were: <0.0001 for Scn2a versus Scn3a, Scn3a versus Scn4a, Scn3a versus Scn8a, Scn3a versus Scn9a; and 0.0104 for Scn4a versus Scn9a. In B, adjusted P values <0.05 were: <0.0001 for Scn1a versus Scn9a; 0.0002 for Scn2a versus Scn9a; 0.0181 for Scn3a versus Scn9a; and 0.0003 for Scn8a versus Scn9a. (C) Example Nav current activation from a WT mouse CC. (D) Example current from a CC from an Nav1.3 KO mouse (typical of 26 Nav1.3 KO CCs) with clear Cav currents and no detectable Nav current. (E) Example current from an Nav1.3 KO CC with a mix of Nav current and Cav current (31 of 57 CCs exhibited at least some rapidly inactivating inward current). Traces in C–E were not leak subtracted. (F) Average peak inward current density (± SD) from 18 WT CCs, 26 Nav1.3 KO cells lacking Nav current (blue), and subtracted Nav current (Fig. S1) from 31 Nav1.3 KO cells. (G) Comparison of Nav current density for WT, Nav1.3 KO cells with detectable Nav current, and then all Nav1.3 KO (n = 57) cells. (H) Comparison of G-V curves for WT and Nav1.3 KO cells (16 cells with >750 pA peak Nav current). Fit parameters: for WT, V0.5 = 22.3 mV, k = 5.3e; for Nav1.3 KO, V0.5 = −17.2 mV, k = 4.9e. From averages of fits to individual cells, for WT, V0.5 = −21.9 ± 2.9 mV, k = 5.8 ± 1.8e; for Nav1.3 KO, V0.5 = −16.8 ± 2.7 mV, k = 4.8 ± 0.7e. V0.5 and k values differ at P = 0.000 based on KS test. (I) Comparison of steady-state inactivation measured from 18 WT cells and 9 Nav1.3 KO cells with Nav currents. Note that the conditioning step duration was 500 ms for Nav1.3 KO cells and 1 s for WT cells. Fits to averaged data yielded, for WT, V0.5 = −49.2 mV, with k = 4.8e, while for Nav1.3 KO cells, V0.5 = −50.0 mV, with k = 5.0e. Averages of fits to individual cells yielded, for WT, V0.5 = −48.9 ± 4.0 mV with k = 5.4 ± 0.7e, while for Nav1.3 cells, V0.5 = −49.9 ± 3.3 mV, with k = 5.3 ± 0.7e. t test P values showed no differences for either V0.5 values, P = 0.510, or k values, P = 0.771.

Nav1.3 (SCN3A) is the major contributor to inward Na+ current in mouse CCs. (A) Each point represents quantitative RT-PCR reactions run in triplicate for a single AM for the indicated Nav channel subunits from mouse AM, with message levels normalized to β-actin. (B) Similar reactions were run on rat AM. In both A and B, at least three AMs were examined for each message species. In A, P values (from ANOVA with Tukey’s post hoc test) <0.05 were: <0.0001 for Scn2a versus Scn3a, Scn3a versus Scn4a, Scn3a versus Scn8a, Scn3a versus Scn9a; and 0.0104 for Scn4a versus Scn9a. In B, adjusted P values <0.05 were: <0.0001 for Scn1a versus Scn9a; 0.0002 for Scn2a versus Scn9a; 0.0181 for Scn3a versus Scn9a; and 0.0003 for Scn8a versus Scn9a. (C) Example Nav current activation from a WT mouse CC. (D) Example current from a CC from an Nav1.3 KO mouse (typical of 26 Nav1.3 KO CCs) with clear Cav currents and no detectable Nav current. (E) Example current from an Nav1.3 KO CC with a mix of Nav current and Cav current (31 of 57 CCs exhibited at least some rapidly inactivating inward current). Traces in C–E were not leak subtracted. (F) Average peak inward current density (± SD) from 18 WT CCs, 26 Nav1.3 KO cells lacking Nav current (blue), and subtracted Nav current (Fig. S1) from 31 Nav1.3 KO cells. (G) Comparison of Nav current density for WT, Nav1.3 KO cells with detectable Nav current, and then all Nav1.3 KO (n = 57) cells. (H) Comparison of G-V curves for WT and Nav1.3 KO cells (16 cells with >750 pA peak Nav current). Fit parameters: for WT, V0.5 = 22.3 mV, k = 5.3e; for Nav1.3 KO, V0.5 = −17.2 mV, k = 4.9e. From averages of fits to individual cells, for WT, V0.5 = −21.9 ± 2.9 mV, k = 5.8 ± 1.8e; for Nav1.3 KO, V0.5 = −16.8 ± 2.7 mV, k = 4.8 ± 0.7e. V0.5 and k values differ at P = 0.000 based on KS test. (I) Comparison of steady-state inactivation measured from 18 WT cells and 9 Nav1.3 KO cells with Nav currents. Note that the conditioning step duration was 500 ms for Nav1.3 KO cells and 1 s for WT cells. Fits to averaged data yielded, for WT, V0.5 = −49.2 mV, with k = 4.8e, while for Nav1.3 KO cells, V0.5 = −50.0 mV, with k = 5.0e. Averages of fits to individual cells yielded, for WT, V0.5 = −48.9 ± 4.0 mV with k = 5.4 ± 0.7e, while for Nav1.3 cells, V0.5 = −49.9 ± 3.3 mV, with k = 5.3 ± 0.7e. t test P values showed no differences for either V0.5 values, P = 0.510, or k values, P = 0.771.

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