Functional cross-talk between phosphorylation and disease-causing mutations in the cardiac sodium channel Nav1.5.

The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Nav1.5 undergoes extensive posttranslational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable posttranslational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semisynthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both wild-type (WT) and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Nav1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity toward the clinically used antiarrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens.

An open state of a voltage-gated sodium channel involving a π-helix and conserved pore-facing asparagine.

Voltage-gated sodium (Nav) channels play critical roles in propagating action potentials and otherwise manipulating ionic gradients in excitable cells. These channels open in response to membrane depolarization, selectively permeating sodium ions until rapidly inactivating. Structural characterization of the gating cycle in this channel family has proved challenging, particularly due to the transient nature of the open state. A structure from the bacterium Magnetococcus marinus Nav (NavMs) was initially proposed to be open, based on its pore diameter and voltage-sensor conformation. However, the functional annotation of this model, and the structural details of the open state, remain disputed. In this work, we used molecular modeling and simulations to test possible open-state models of NavMs. The full-length experimental structure, termed here the α-model, was consistently dehydrated at the activation gate, indicating an inability to conduct ions. Based on a spontaneous transition observed in extended simulations, and sequence/structure comparison to other Nav channels, we built an alternative π-model featuring a helix transition and the rotation of a conserved asparagine residue into the activation gate. Pore hydration, ion permeation, and state-dependent drug binding in this model were consistent with an open functional state. This work thus offers both a functional annotation of the full-length NavMs structure and a detailed model for a stable Nav open state, with potential conservation in diverse ion-channel families.

Modulation of Pore Opening of Eukaryotic Sodium Channels by π-Helices in S6.

Voltage-gated sodium channels are heterotetrameric sodium selective ion channels that play a central role in electrical signaling in excitable cells. With recent advances in structural biology, structures of eukaryotic sodium channels have been captured in several distinct conformations corresponding to different functional states. The secondary structure of the pore lining S6 helices of subunits DI, DII, and DIV has been captured with both short π-helix stretches and in fully α-helical conformations. The relevance of these secondary structure elements for pore gating is not yet understood. Here, we propose that a π-helix in at least DI-S6, DIII-S6, and DIV-S6 results in a fully conductive state. On the other hand, the absence of π-helix in either DI-S6 or DIV-S6 yields a subconductance state, and its absence from both DI-S6 and DIV-S6 yields a nonconducting state. This work highlights the impact of the presence of a π-helix in the different S6 helices of an expanded pore on pore conductance, thus opening new doors toward reconstructing the entire conformational landscape along the functional cycle of Nav Channels and paving the way to the design of state-dependent modulators.

An α-π transition in S6 shapes the conformational cycle of the bacterial sodium channel NavAb.

Voltage-gated sodium channels play an important role in electrical signaling in excitable cells. In response to changes in membrane potential, they cycle between nonconducting and conducting conformations. With recent advances in structural biology, structures of sodium channels have been captured in several distinct conformations, which are thought to represent different functional states. However, it has been difficult to capture the intrinsically transient open state. We recently showed that a proposed open state of the bacterial sodium channel NavMs was not conductive and that a conformational change involving a transition to a π-helix in the pore-lining S6 helix converted this structure into a conducting state. However, the relevance of this structural feature in other sodium channels, and its implications for the broader gating cycle, remained unclear. Here, we propose a comparable open state of another class of bacterial channel from Aliarcobacter butzleri (NavAb) with characteristic pore hydration, ion permeation, and drug binding properties. Furthermore, we show that a π-helix transition can lead to pore opening and that such a conformational change blocks fenestrations in the inner helix bundle. We also discover that a region in the C-terminal domain can undergo a disordering transition proposed to be important for pore opening. These results support a role for a π-helix transition in the opening of NavAb, enabling new proposals for the structural annotation and drug modulation mechanisms in this important sodium channel model.

Subtype-specific responses of hKv7.4 and hKv7.5 channels to polyunsaturated fatty acids reveal an unconventional modulatory site and mechanism.

The KV7.4 and KV7.5 subtypes of voltage-gated potassium channels play a role in important physiological processes such as sound amplification in the cochlea and adjusting vascular smooth muscle tone. Therefore, the mechanisms that regulate KV7.4 and KV7.5 channel function are of interest. Here, we study the effect of polyunsaturated fatty acids (PUFAs) on human KV7.4 and KV7.5 channels expressed in Xenopus oocytes. We report that PUFAs facilitate activation of hKV7.5 by shifting the V50 of the conductance versus voltage (G(V)) curve toward more negative voltages. This response depends on the head group charge, as an uncharged PUFA analogue has no effect and a positively charged PUFA analogue induces positive V50 shifts. In contrast, PUFAs inhibit activation of hKV7.4 by shifting V50 toward more positive voltages. No effect on V50 of hKV7.4 is observed by an uncharged or a positively charged PUFA analogue. Thus, the hKV7.5 channel's response to PUFAs is analogous to the one previously observed in hKV7.1-7.3 channels, whereas the hKV7.4 channel response is opposite, revealing subtype-specific responses to PUFAs. We identify a unique inner PUFA interaction site in the voltage-sensing domain of hKV7.4 underlying the PUFA response, revealing an unconventional mechanism of modulation of hKV7.4 by PUFAs.

In order to carry out their roles in the body, cells need to send and receive electrical signals. They can do this by allowing ions to move in and out through dedicated pore-like structures studded through their membrane. These channels are specific to one type of ions, and their activity ­ whether they open or close ­ is carefully controlled. In humans, defective ion channels are associated with conditions such as irregular heartbeats, epileptic seizures or hearing loss. Research has identified molecules known as polyunsaturated fatty acids as being able to control the activity of certain members of the K_V7 family of potassium ion channels. The K_V7.1 and K_V7.2/7.3 channels are respectively present in the heart and the brain; K_V7.4 is important for hearing, while K_V7.5 plays a key role in regulating muscle tone in blood vessels. Polyunsaturated fatty acids can activate K_V7.1 and K_V7.2/7.3 but their impact on K_V7.4 and K_V7.5 remains unclear. Frampton et al. explored this question by studying human K_V7.4 and K_V7.5 channels expressed in frog egg cells. This showed that fatty acids activated K_V7.5 (as for K_V7.1 and K_V7.2/7.3), but that they reduced the activity of K_V7.4. Closely examining the structure of K_V7.4 revealed that the fatty acids were binding to a different region compared to the other K_V7 channels. When this site was made inaccessible, fatty acids increased the activity of K_V7.4, just as for the rest of the family. These results may help to understand the role of polyunsaturated fatty acids in the body. In addition, knowing how these molecules interact with channels in the same family will be useful for optimising a drug's structure to avoid side effects. However, further research will be needed to understand the broader impact in a more complex biological organism.

Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity.

Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD's localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of INa by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.

The use of local structural similarity of distant homologues for crystallographic model building from a molecular-replacement solution.

The performance of automated protein model building usually decreases with resolution, mainly owing to the lower information content of the experimental data. This calls for a more elaborate use of the available structural information about macromolecules. Here, a new method is presented that uses structural homologues to improve the quality of protein models automatically constructed using ARP/wARP. The method uses local structural similarity between deposited models and the model being built, and results in longer main-chain fragments that in turn can be more reliably docked to the protein sequence. The application of the homology-based model extension method to the example of a CFA synthase at 2.7 Šresolution resulted in a more complete model with almost all of the residues correctly built and docked to the sequence. The method was also evaluated on 1493 molecular-replacement solutions at a resolution of 4.0 Šand better that were submitted to the ARP/wARP web service for model building. A significant improvement in the completeness and sequence coverage of the built models has been observed.