Crystallographic basis for calcium regulation of sodium channels

Abstract

Voltage-gated sodium channels underlie the rapid regenerative upstroke of action potentials and are modulated by cytoplasmic calcium ions through a poorly understood mechanism. We describe the 1.35 Å crystal structure of Ca(2+)-bound calmodulin (Ca(2+)/CaM) in complex with the inactivation gate (DIII-IV linker) of the cardiac sodium channel (Na(V)1.5). The complex harbors the positions of five disease mutations involved with long Q-T type 3 and Brugada syndromes. In conjunction with isothermal titration calorimetry, we identify unique inactivation-gate mutations that enhance or diminish Ca(2+)/CaM binding, which, in turn, sensitize or abolish Ca(2+) regulation of full-length channels in electrophysiological experiments. Additional biochemical experiments support a model whereby a single Ca(2+)/CaM bridges the C-terminal IQ motif to the DIII-IV linker via individual N and C lobes, respectively. The data suggest that Ca(2+)/CaM destabilizes binding of the inactivation gate to its receptor, thus biasing inactivation toward more depolarized potentials.

Fig. 1.

Crystal structure of the Ca²⁺/CaM–Na_V1.5 DIII-IV linker complex. (A) Ca²⁺/CaM C lobe bound to the DIII-IV linker in the open/extended conformation. A 90° rotated view highlights charged residues (K1492, K1493, K1499, K1500) facing away from the binding pocket and Y1494 buried into the C-lobe pocket. (B) A bottom-up view of the inactivation gate interacting with the C lobe, with the surface of calmodulin colored according to amino acid type (yellow, hydrophobic; green, neutral; red, acidic; blue, basic), with Y1494 facing away from the viewer. Select residues are labeled to orient the reader. Boxed residues are for the DIII-IV linker, with targets for disease mutations in bold. (C) Side view of the interaction highlights the details of the hydrophobic pocket that defines the Y1494 interaction with the CaM C lobe.

Fig. 2.

Molecular determinants for CaM binding to the DIII-IV linker. (A) Sequence alignment of nine human Na_V isoforms, with deviations from the Na_V1.5 sequence highlighted in purple. In the Na_V1.5 sequence, residues highlighted in yellow mark physiological mutations for LQT3 and Brugada syndromes. The highly conserved double-glutamate motif that reduces the affinity of CaM to the DIII-IV linker is boxed. (B) Binding displayed by the DIII-IV linker construct used for crystallization; Ca²⁺/CaM (1.5 mM) to 1491–1522 (150 μM). (C) (Top) Raw heats from the Ca²⁺/N lobe (1 mM) titration to a premixed Ca²⁺/C lobe (200 μM) plus DIII-IV (100 μM). Heats were not distinguishable compared with background heats, thus demonstrating that the affinity was below the detection limit. (Middle and Bottom) Raw and integrated heats for the titration of Ca²⁺/C lobe (1 mM) to a premixed Ca²⁺/N lobe (200 μM) plus DIII-IV (100 μM). (D) The change in free energy compared with Ca²⁺/CaM binding to the full-length inactivation gate. A positive and negative ΔΔG correlates to higher and lower affinity, respectively, for the indicated construct compared with the WT DIII-IV linker. Thermodynamic parameters for each ITC can be found in Table S2.

Fig. 3.

The DIII-IV linker is the molecular endpoint for Ca²⁺ regulation of the cardiac sodium channels. Steady-state inactivation relationships in 0, 300 nM, and 10 μM free Ca²⁺ in the recording pipette for WT (A), M1498A (B), and EE/AA (C) channels with representative normalized currents (Insets). EE/AA channels display enhanced sensitivity to 300 nM Ca²⁺, whereas WT channels do not. Inorm_Na, the amount of current available during a 20-ms test-pulse to −20 mV after a 500-ms pre-pulse to the indicated voltage. (D) Ca²⁺ dependence of the shift of steady-state inactivation for both WT and EE/AA channels. (E) EE/AA channels have slowed inactivation in the presence of Ca²⁺. Time course of fast inactivation [τ_inact (ms)] from currents produced by a depolarization from −120 mV to the indicated voltage (Insets are from a −40-mV step) and fit with a single exponential. Electrophysiological parameters for both (D) and (E) can be found in Tables S5 and S6. (F) Affinity of CaM for the DIII-IV linker at physiological Ca²⁺ concentrations. Thermodynamic parameters are shown in Table S3. [Scale bars (A and E), 5 ms.]

Fig. 4.

Thermodynamic basis for Ca²⁺-dependent interactions between CaM, the inactivation gate, and the C terminus. ITC titrations of the indicated constructs (Insets) in the raw ITC data trace (Top and Middle) and integrated heats of the measured interaction (Bottom). (A) Apo-N lobe (250 μM) to IQ domain (25 μM) (Top) (no binding detected), with apo-C lobe (250 μM) to IQ domain (25 μM) (Middle and Bottom), K_d = 0.34 μM. (B) Ca²⁺/CaM (500 μM) to CTD, no IQ (50 μM) (Top) showing no binding, with Ca²⁺/CaM (1 mM) to CTD (80 μM) titration shown below, K_d = 6.45 μM. (C) Ca²⁺/C lobe (500 μM) to CTD (50 μM) (Top), K_d = 7.6 μM, with Ca²⁺/N lobe (500 μM) to CTD (50 μM) (Middle), K_d = 2.53 μM, shown below with the integration of Ca²⁺/C lobe (in blue) and Ca²⁺/N lobe (in green). The integration of Ca²⁺/C lobe into a mixture of CTD with the Ca²⁺/N lobe is shown (Bottom) in red. Thermodynamic parameters can be found in Table S4.

Fig. 5.

Mechanism of Ca²⁺ regulation of voltage-gated sodium channels. (Upper) A Ca²⁺-free scenario where a resident apo-CaM molecule is bound to the C-terminal IQ motif via the C lobe of CaM. In this conformation, neither CaM lobe interacts with the DIII-IV linker and inactivation gating is left unaffected. (Lower) Ca²⁺ ions (shown as black circles) bind to CaM and promote lobe switching whereby the N lobe now occupies the C-terminal IQ motif and the C lobe binds to the DIII-IV linker, where it effects equilibrium inactivation gating by destabilizing the inactivated state of the channel.