Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

Abstract

In cardiomyocytes, Na_V1.5 channels mediate initiation and fast propagation of action potentials. The Ca²⁺-binding protein calmodulin (CaM) serves as a de facto subunit of Na_V1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the Na_V1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a "gain-of-function" defect, and Brugada syndrome, a "loss-of-function" phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on Na_V1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca²⁺-free CaM preassociation with Na_V1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent Na_V openings. Mechanistically, these effects arise from a CaM-dependent switch in the Na_V inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant Na_V1.5, local Ca²⁺ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of Na_V1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of Na_V1.5 channelopathies.

Keywords: Brugada syndrome; Nav1.5; calmodulin; ion channels; long QT syndrome.

Fig. 1.

Absence of dynamic Ca²⁺/CaM effects on WT Na_V1.5 SSI. (A, Left) Structure of Na_V1.5 transmembrane domain (6UZ3) (70) juxtaposed with that of Na_V1.5 CT–apoCaM complex (4OVN) (28). (Right) Arrhythmia-linked CT mutations highlighted in Na_V1.5 CT–apoCaM structure (LQTS3, blue; BrS, magenta; mixed syndrome, purple). (B) Dynamic Ca²⁺-dependent changes in Na_V1.5 SSI probed using Ca²⁺ photouncaging. Na currents specifying h_∞ at ∼100 nM (Left) and ∼4 μM Ca²⁺ step (Right). (C) Population data for Na_V1.5 SSI under low (black, Left) versus high (red, Right) intracellular Ca²⁺ reveal no differences (P = 0.55, paired t test). Dots and bars are mean ± SEM (n = 8 cells). (D) FRET two-hybrid analysis of Cerulean-tagged apoCaM interaction with various Venus-tagged Na_V1.5 CT (WT, black; IQ/AA, red; S[1904]L, blue). Each dot is FRET efficiency measured from a single cell. Solid line fits show 1:1 binding isotherm.

Fig. 2.

Disruption of apoCaM preassociation diminishes peak P_O of Na_V1.5. (A) SSI (h_∞) curve for WT Na_V1.5 measured under endogenous (black), low (red), and high (blue) CaM levels. Low CaM levels were attained by overexpressing a CaM chelator. High CaM levels were attained by overexpressing recombinant CaM. (Inset) Voltage protocol. (B and C) Format as in A but for Na_V1.5 IQ/AA (B) and S[1904]L (C) mutants. (D) Exemplar traces showing stochastic records of channel openings from a one-channel patch of Na_V1.5 WT evoked in response to a depolarizing pulse to −10 mV. Solid line denotes zero-current baseline, and channel openings are downward deflections to the unitary current level (dashed line). (E) Ensemble average P_O waveform from a single patch (147 sweeps) reveals a P_O/peak ∼0.5. (F) Population P_O/peak values are plotted as block dots and bars (mean ± SEM) as a function of the activating test pulse potential (n = 7 patches, 1,768 sweeps/voltage). The black line is the single Boltzmann fit for P_O/peak–V relation with parameters: P_O,max = 0.48; V_1/2 = −36 mV, and slope factor (SF) = 9.5. (G–I) Format as in D–F but for Na_V1.5 IQ/AA. (H) Ensemble P_O average shown from a single patch with 120 sweeps. (I) Population data of Na_V1.5 IQ/AA P_O/peak–V relationship is shown by pale red dots (mean ± SEM, n = 5, 1,088 sweeps/voltage) and red line fit. Boltzmann fit parameters: P_O,Max = 0.21; V_1/2 = −45 mV; SF = 9.5. Gray line is the WT fit reproduced from F. (J–L) Format as in D–F but for NaV1.5 S[1904]L. (K) Ensemble P_O average obtained from 254 sweeps in a single patch. (L) Population data for Na_V1.5 S[1904]L P_O/peak–V relationship is shown by pale red dots (mean ± SEM, n = 6, 1,365 sweeps/voltage) and red line fit. Boltzmann fit parameters for Na_V1.5 S[1904]L: P_O,Max = 0.26; V_1/2 = −45 mV; SF = 9.5. Gray line is the WT fit reproduced from F. Statistical analysis: two-way ANOVA followed by post hoc Tukey’s multiple comparisons test, **P < 0.01 with WT channels as reference.

Fig. 3.

ApoCaM binding tunes maximal P_O of mutant Na_V1.5 with reduced CaM binding. (A and B) Exemplar stochastic single-channel recordings of Na_V1.5 IQ/AA under CaM-overexpressed (A) and CaM-depleted (B) conditions. (C) Ensemble P_O average of Na_V1.5 IQ/AA from a single patch under CaM-overexpressed (blue, 130 sweeps) and CaM-depleted (red, 276 sweeps) conditions. (D) Population Na_V1.5 IQ/AA P_O/peak–V relationship under CaM-overexpressed (blue dots and fit, n = 5, 539 sweeps/voltage) and CaM-depleted (red dots and fit, n = 7, 1,249 sweeps/voltage) conditions. Gray is the Na_V1.5 IQ/AA fit under endogenous CaM levels reproduced from Fig. 2I. All dots and bars are mean ± SEM. Boltzmann fit parameters for CaM-overexpressed condition (blue): P_O,max = 0.45; V_1/2 = −38 mV; SF = 9.5. Boltzmann fit parameters for CaM-depleted condition (red): P_O,max = 0.23; V_1/2 = −40 mV; SF = 9.5. Statistical analysis: two-way ANOVA followed by post hoc Tukey’s multiple comparisons test, ***P < 0.001 comparing the CaM overexpression with the CaM-depleted condition. (E and F) Format as in A–D but for Na_V1.5 S[1904]L mutant. (G) Ensemble P_O average of Na_V1.5 S[1904]L from a single patch under CaM-overexpressed (blue, 120 sweeps) and CaM-depleted (red, 218 sweeps) conditions. (H) Population Na_V1.5 S[1904]L P_O/peak–V relationship under CaM-overexpressed (blue dots and fit, n = 11, 2,496 sweeps/voltage) and CaM-depleted (red dots and fit, n = 8, 1,568 sweeps/voltage) conditions. Gray is the Na_V1.5 S[1904]L fit under endogenous CaM levels reproduced from Fig. 2L. Boltzmann fit parameters for CaM-overexpressed condition (blue): P_O,Max = 0.5; V_1/2 = −43 mV; SF = 9.5. Boltzmann fit parameters for CaM-depleted condition (red): P_O,Max = 0.18; V_1/2 = −45 mV; SF = 9.5. Statistical analysis: two-way ANOVA followed by post hoc Tukey’s multiple comparisons test, **P < 0.01 and ***P < 0.001 comparing the CaM overexpression with the CaM-depleted condition.

Fig. 4.

Elementary mechanisms underlying CaM regulation of Na_V1.5. (A and B) FL distributions for Na_V1.5 WT (gray shaded area) and Na_V1.5 IQ/AA mutant under CaM-overexpressed (pale blue shaded area) and CaM-depleted (rose shaded area) conditions. FL denotes the probability that the first opening occurred at time < t. Solid lines are fits of FL distributions generated by the model shown in Fig. 4I. The difference in FL distributions for Na_V1.5 IQ/AA at low versus high CaM levels are statistically significant (P < 0.001, Kolmogorov–Smirnov [KS] test). (C and D) Histograms of OD distribution which correspond to the time spent in the open state for WT (gray shaded area, C) and IQ/AA under low (rose shaded area, D) and high (pale blue shaded area, D) CaM conditions. Solid lines are fits of OD distributions generated by the model shown in Fig. 4I. The OD distribution of IQ/AA is significantly prolonged under low CaM levels compared to CaM overexpression (D, P < 0.001, KS test). (E and F) Distribution of the number of openings per sweep for WT (black) and IQ/AA mutant channels at low (red) and high (blue) levels of ambient CaM. ***P < 0.001, test of proportion for comparing fraction of blank sweeps. (G and H) Conditional open probability (P_OO(t)) for WT (gray shaded area) and IQ/AA at low (rose shaded area) or high (pale blue shaded area) CaM levels. Solid lines are reproduction of the fits of OD distributions in C and D. For Na_V1.5 IQ/AA, P_OO(t), distributions at low versus high CaM are statistically different (P < 0.001, KS test). (I) Summary of the effect of CaM on Na_V1.5 gating. Absent CaM, channels preferentially inactivate from the closed state. Upon binding CaM, inactivation proceeds preferentially from the open state (α: rate of transition from the closed to open state, β: rate of transition from the open to closed state, k_OI: rate of inactivation from the open state, k_CI: rate of inactivation from the closed state, k_OC: rate of channel closure, and k_CO: rate of channel opening).

Fig. 5.

ApoCaM interaction with Na_V1.5 tunes likelihood for persistent channel openings. (A, Top) Representative multichannel record from Na_V1.5 WT shows rapid activation and inactivation followed by rare openings in the late phase following 50 ms of depolarization (gray shaded region). (Inset) Enlarged late phase to better visualize Na_V1.5 openings. (Bottom) Normalized ensemble average open probability (n = 16, 875 sweeps). (Inset) Enlarged normalized open probability in the late phase. (B and C) Format as in A Top but for Na_V1.5 WT channels recorded under CaM-depleted (B) and CaM-overexpressed (C) conditions. (D–I) Format as in A–C but for Na_V1.5 IQ/AA (D–F) and S[1904]L (G–I) mutants. (J) Quantification and population data for persistent channel openings for Na_V1.5 WT, IQ/AA, and S[1904]L under different CaM concentrations (A–I). For each condition, a normalized ensemble open probability ${\widehat{P}}_O$ is calculated by averaging many sweeps (SI Appendix, Fig. S7). R_persist is the average open probability ${\widehat{P}}_O$ in the late phase (gray shaded regions in A–I) normalized by the peak ${\widehat{P}}_O$. Each bar and error, mean ± SEM. Statistical analysis: one-way ANOVA followed by Tukey multiple comparisons test. ***P < 0.001, **P < 0.01, *P < 0.05 compared to WT at endogenous CaM levels; ^##P < 0.01 versus IQ/AA at endogenous CaM level; and ^††P < 0.01 when compared to S1904L at endogenous CaM levels. (K) Correlation of R_persist versus P_O/peak under different CaM concentrations shows a linear relationship.

Fig. 6.

Dynamic Ca²⁺ fluctuations tune persistent Na_V channel openings. (A) FRET two-hybrid analysis probes the interaction of Venus-tagged Na_V1.5 CT with Cerulean-tagged CaM at high Ca²⁺ levels. Black dots and fit, WT; red dots and fit, IQ/AA mutant; blue dots and fit, S[1904]L mutant. (B) Bar graph summary compares K_a,EFF = 1/K_d,EFF for Ca²⁺ versus apoCaM binding to WT Na_V1.5 as well as IQ/AA and S[1904]L mutant. (C) Exemplar multichannel stochastic record from cell-attached recordings of Na_V1.5 WT coexpressed with Ca_V2.1. Na_V1.5 openings are evoked using the −35 mV voltage pulse. Ensuing +15 mV voltage pulse elicits Ca²⁺ channel openings (rose shaded area). A subsequent pulse to −35 mV is used to identify Ca²⁺-dependent changes in persistent Na_V channel openings. (D) Normalized ensemble average ${\widehat{P}}_O$ waveform is computed from 293 sweeps (six patches). A comparison of the ${\widehat{P}}_O$ waveform before (black) and after (red) Ca²⁺ pulse reveals minimal differences in the late Na current. (E–H) Exemplar multichannel recordings and ensemble average ${\widehat{P}}_O$ waveforms of Na_V1.5 IQ/AA (E and F) or S[1904]L (G and H) coexpressed with Ca_V2.1. Format same as C and D. Data obtained from 342 sweeps (seven patches) for Na_V1.5 IQ/AA and 264 sweeps (seven patches). (I) Paired dot plot summarizes changes in R_persist before (black dot, gray bar) and after (red dot, rose bar) Ca²⁺ influx. Only the IQ/AA mutant showed a consistent reduction in the late current following Ca²⁺ entry (**P < 0.01 by paired t test).

Fig. 7.

In-depth analysis suggests a distinct open state associated with late Na current. (A) Pale blue shaded area shows the conditional OD histogram for Na_V1.5 WT channel opening in the late phase (after 50 ms depolarization), which follows a multiexponential decay. The gray solid line is the OD fit for the WT channel opening in the early phase (reproduced from SI Appendix, Fig. S4E). The red solid line is a scaled OD fit for the IFM/IQM inactivation-deficient mutant opening (B). The blue solid line is a fit for the late-phase conditional OD by a weighted sum of the early phase and IFM/IQM OD distributions. (B) OD histogram (pale red shaded area) and fit (solid red line) of the IFM/IQM mutant opening. (C–F) Format as in A but for Na_V1.5 IQ/AA or S[1904]L mutants at both low and high ambient CaM levels. Gray solid lines are OD fits for the respective mutant channel openings in the early phase (reproduced from SI Appendix, Figs. S4F and S5E). In all cases, the conditional OD distributions for late channel openings are multiexponential, with the slow component matching the OD distribution of inactivation-deficient (IFM/IQM) channels. (G) Summary of multifaceted modulation of Na_V1.5 by CaM and potential molecular conformational changes. With CaM bound, the carboxy-terminus (CT) interacts with the III-IV linker and prevents premature translocation for the “IFM” inactivation particle to its receptor site near the S6 domain. Following voltage depolarization and channel opening, the III-IV linker may release from the CT and thereby trigger channel inactivation. Devoid of apoCaM, allosteric changes may cause the III-IV linker to be released from the CT. This change could result in premature translocation of the “IFM” motif to the S6 receptor site, manifesting as enhanced closed-state inactivation. Alternatively, with a low likelihood, the “IFM” motif may altogether fail to reach its receptor site, resulting in persistent channel openings.