A Double Tyrosine Motif in the Cardiac Sodium Channel Domain III-IV Linker Couples Calcium-dependent Calmodulin Binding to Inactivation Gating

Abstract

Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr 1494 and Tyr 1495 ) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.

Crystallographic basis for calcium regulation of sodium channels

“…The DIII-IV linker residue M1498 has a large contact area with CaM with 107 Å 2 out of 118 Å 2 buried (Fig. 1B), suggesting it could contribute to Ca 2+ /CaM binding and regulation of Na V s. Previous ITC experiments have shown that the interaction requires Ca 2+ and is supported preferentially by the C lobe (K d 19 μM versus ∼600 μM for the N lobe) (9). In addition, competition experiments whereby the C lobe is already bound to the DIII-IV linker show that the residual affinity of the N lobe is undetectable (Fig.…”

“…2B; K d 0.35 μM vs. 2.98 μM for WT). The energetic impact of single Y1494A or Y1495A mutations on the WT or 1491-1522 constructs results in enthalpic penalties of 5 kcal/mol and 3 kcal/mol, respectively, which were matched with an equal gain in entropy, leaving the overall affinities unchanged (Table S2) (9). Given the identical energetic signature and enthalpy-entropy compensations, the interactions with Ca 2+ /CaM are likely to be the same in 1491-1522 and full-length DIII-IV linkers.…”

“…S1A). Previously, Y1494 was identified as a residue of importance, as alanine substitution alters Ca 2+ /CaM binding energetics (as measured by ITC) and uncouples Ca 2+ regulation of channel inactivation (9). The DIII-IV linker residue M1498 has a large contact area with CaM with 107 Å 2 out of 118 Å 2 buried (Fig.…”

“…Ca 2+ levels fluctuate during the excitation-contraction cycle in cardiac myocytes, and the total cytoplasmic Ca 2+ concentration for halfmaximal activation of contraction can reach ∼70 μM (2). Although it is known that the CaM-DIII-IV linker interaction requires Ca 2+ for binding (9,10), the dynamic range of Ca 2+ levels, and more importantly the lower limit over which CaM can bind the inactivation gate, has remained unexplored. Here the data show that the interaction maintains affinity at low free Ca 2+ levels (∼K d of 25.3 μM in 100 nM free Ca 2+ ) (Fig.…”

“…Consequently, their cytoplasmic levels oscillate between nanomolar and micromolar levels with each excitation-contraction cycle (2). Sodium channel steady-state inactivation, a process that controls channel availability at a given transmembrane potential, is modulated through interactions with Ca 2+ and calmodulin (CaM) (3)(4)(5)(6)(7)(8)(9)(10) can bind directly to the Na V "inactivation gate" (9, 10), a highly conserved ∼50-amino acid cytoplasmic linker connecting channel domains III and IV (DIII-IV linker) that contributes to the fastinactivated state of the channel (14)(15)(16). Thus, individual molecular players of Ca 2+ regulation have been identified but no crystallographic information is available detailing how the different Ca 2+ /CaM binding regions cooperate, and no consensus has been reached on how binding events in the C terminus of the channel are coupled to the conformational changes that underlie channel inactivation.…”