Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin

Potet, F; Chagot, Benjamin; Anghelescu, Mircea; Viswanathan, Prakash C.; Stepanovic, Svetlana Z.; Kupershmidt, Sabina; Chazin, Walter J.; Balser, Jeffrey R.

doi:10.1074/jbc.m806871200

Cited by 75 publications

(103 citation statements)

References 40 publications

(54 reference statements)

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“…Taken together, the data support a model shown in Fig. 5 where in low Ca 2+ (apo-CaM), the C lobe associates with the IQ domain, and neither CaM lobe is able to interact with the DIII-IV linker (9,10). In the presence of Ca 2+ , CaM bridges two cytoplasmic segments, with Ca 2+ /N lobe bound to the CTD and Ca 2+ /C lobe bound to the DIII-IV linker.…”

Section: Discussionsupporting

confidence: 68%

“…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.…”

Section: Diii-iv Linker Is the Physiological Endpoint For Camentioning

confidence: 99%

“…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.…”

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confidence: 99%

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Crystallographic basis for calcium regulation of sodium channels

Sarhan

Tung²,

Petegem³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

125

140

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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.crystallography | patch-clamp electrophysiology | structural biology | cardiac arrhythmia V oltage-gated sodium channels (Na V s) support excitability in the cardiovascular and nervous systems, where they contribute to the rhythm and rate of action potentials. These large (∼220-kDa) transmembrane protein complexes are expressed at a high density in excitable cells, where they conduct large macroscopic inward sodium currents. These channels are exquisitely sensitive to subtle changes in the transmembrane potential, and modest alterations in channel gating can fine-tune or disorder electrical signaling at the organ and systemic level. The α-subunit of the channel contains cytoplasmic amino and carboxyl termini and is composed of four homologous transmembrane domains (DI-DIV) that are connected by intracellular linkers. Each domain contains voltage-sensing (S1-S4) and pore-forming (S5 and S6) domains that form the selectivity filter and putative activation gates. A crystal structure of a bacterial Na V was recently described (1) showing a similar overall fold compared with potassium channels. However, this bacterial variant is homotetrameric, and seems to lack a conserved fast-inactivation mechanism. As such, it has no homology with several relevant domains in mammalian Na V channels, and no crystal structure of any eukaryotic Na V region has yet been reported.Calcium ions (Ca 2+ ) are universal second messengers, and in the heart they form the electrochemical link between plasma membrane depolarization and myocyte contraction. 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) (3-10). The mechanistic details of Ca 2+ modulation of sodium channel inactivation are sparse, but the C-terminal region of the cha...

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Section: Discussionsupporting

confidence: 68%

Section: Diii-iv Linker Is the Physiological Endpoint For Camentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Crystallographic basis for calcium regulation of sodium channels

Sarhan

Tung²,

Petegem³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

125

140

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“…Although the precise mechanistic details of this modulation remain speculative, the C-terminal region contains EF-hand like domains and an IQ motif that dynamically bind Ca 2ϩ and Ca 2ϩ /CaM, respectively, and mutations in these regions affect both calcium sensitivity and inactivation gating (6,11,(13)(14)(15)(16). Additionally, recent evidence suggests that CaM can bind directly to the DIII-DIV linker, a possibility that has been suggested previously in the context of an "inactivation complex" (14,17), therefore providing yet another pathway for Ca 2ϩ /CaM regulation of channel gating (18). Interestingly, introduced mutations into the putative Ca 2ϩ /CaM binding regions in the DIII-IV linker or the IQ motif do not explicitly interfere with calcium modulation of channel inactivation (16,18), suggesting that Ca 2ϩ /CaM binding to the channel per se may not play a pivotal role in sensitizing the inactivation pro-cess to calcium.…”

mentioning

confidence: 99%

“…Additionally, recent evidence suggests that CaM can bind directly to the DIII-DIV linker, a possibility that has been suggested previously in the context of an "inactivation complex" (14,17), therefore providing yet another pathway for Ca 2ϩ /CaM regulation of channel gating (18). Interestingly, introduced mutations into the putative Ca 2ϩ /CaM binding regions in the DIII-IV linker or the IQ motif do not explicitly interfere with calcium modulation of channel inactivation (16,18), suggesting that Ca 2ϩ /CaM binding to the channel per se may not play a pivotal role in sensitizing the inactivation pro-cess to calcium. Alternatively, it is possible that CaM binding plays a central role in calcium regulation of inactivation through yet undetermined binding scenarios.…”

mentioning

confidence: 99%

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

Sarhan

Petegem

Ahern

2009

Journal of Biological Chemistry

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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.

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Ventricular voltage‐gated ion channels: Detection, characteristics, mechanisms, and drug safety evaluation

Chen

Wang

et al. 2021

Clinical & Translational Med

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Cardiac voltage‐gated ion channels (VGICs) play critical roles in mediating cardiac electrophysiological signals, such as action potentials, to maintain normal heart excitability and contraction. Inherited or acquired alterations in the structure, expression, or function of VGICs, as well as VGIC‐related side effects of pharmaceutical drug delivery can result in abnormal cellular electrophysiological processes that induce life‐threatening cardiac arrhythmias or even sudden cardiac death. Hence, to reduce possible heart‐related risks, VGICs must be acknowledged as important targets in drug discovery and safety studies related to cardiac disease. In this review, we first summarize the development and application of electrophysiological techniques that are employed in cardiac VGIC studies alone or in combination with other techniques such as cryoelectron microscopy, optical imaging and optogenetics. Subsequently, we describe the characteristics, structure, mechanisms, and functions of various well‐studied VGICs in ventricular myocytes and analyze their roles in and contributions to both physiological cardiac excitability and inherited cardiac diseases. Finally, we address the implications of the structure and function of ventricular VGICs for drug safety evaluation. In summary, multidisciplinary studies on VGICs help researchers discover potential targets of VGICs and novel VGICs in heart, enrich their knowledge of the properties and functions, determine the operation mechanisms of pathological VGICs, and introduce groundbreaking trends in drug therapy strategies, and drug safety evaluation.

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Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin

Cited by 75 publications

References 40 publications

Crystallographic basis for calcium regulation of sodium channels

Crystallographic basis for calcium regulation of sodium channels

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

Ventricular voltage‐gated ion channels: Detection, characteristics, mechanisms, and drug safety evaluation

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