Voltage-gated sodium channels (Nav channels) play an essential role in nerve impulse conduction in excitable cells. Thus, these channels are involved in several neurological and muscular disorders. Understanding their mechanism of functioning  is essential for designing drugs targeting them. These are tetrameric membrane proteins that selectively transport sodium ions across the membrane. They regulate ion flow by cycling through three main functional states - resting state, open state, and inactivated state. Structural biology techniques have captured Nav channels in several functional states. However, most of the structures are captured in the inactivated state. Although it is quite challenging to capture the open state experimentally because of its transient nature,  several structures of bacterial and eukaryotic Nav channels have been captured in the putative open state. However, a rigorous functional annotation of these open-state structures awaits. 

I performed molecular dynamics simulations to show that the experimental bacterial Nav channels captured in the putative open state, the pore was dehydrated and had a high free energy barrier for ion/drug permeation suggesting that these structures do not correspond to a functional open state. The pore-lining helices of these channels are 𝛼 helical. Sequence/structure conservation analysis showed the possibility of 𝜋-helices in the pore-lining helices. Introducing 𝜋-helices in the middle of these pore-lining helices hydrated the pore and removed the free energy barrier for ion/drug permeation. The 𝜋-helices might also be relevant for pore opening as they dehydrate the peripheral cavities/reduce the interactions between the hydrophobic pore-lining residues and hence allow the opening of the hydrophobic pore. Additionally, I also determined a disordered region in the C-terminal domain which is known to be relevant to pore opening.

I also studied the effect of 𝜋-helices on drug access and binding to sodium channels.  I found that 𝜋-helices in the bacterial Nav channel blocked the fenestrations irrespective of the pore diameter thus inhibiting drug access through the fenestrations. Exploring further on drug binding, I investigated lidocaine binding to different functional states which revealed that the drug binds in different orientations and positions across the functional states. This implies that there might be a change in the lidocaine-binding affinity as the channel cycles through different functional states. I also investigated the drug binding site and access pathway of cannabidiol in sodium channels and the effect of cannabidiol on membrane properties. Our computational results were complemented by experimental results. Molecular dynamics simulations suggest that cannabidiol does not affect the membrane rigidity and causes an ordering of the membrane methylenes, which is in excellent agreement with the NMR results. Mutagenesis experiments show that cannabidiol blocks the pore by interacting with a phenylalanine residue which is in good agreement with our docking results. Adiabatic biased molecular dynamics simulations were performed to confirm the pathway for CBD to reach the pore is through the fenestrations in the ion channel. 

The idea of investigating the relevance of 𝜋-helices in pore-lining helices was extended to eukaryotic Nav channels as well. Eukaryotic channels are heterotetrameric, so the pore lining helices of different subunits might contribute differently to the channel function. I concluded that increasing the number of 𝜋-helices not only increased the pore hydration and ion conductance but also reduced the barrier for ion permeation. 𝜋-helices in pore-lining helices of subunit-I and subunit-IV in an expanded pore are essential for a functional open state.

Putting the above results together, I show that the bacterial experimental structures initially proposed to represent open states might correspond instead to inactivated states. In eukaryotes, the experimental structure initially proposed to represent the open state corresponds to a sub-conductance open state. Thus, I propose that a 𝜋 to 𝛼 helix transition and vice-versa might be relevant to the gating of Nav channels. By showing these results I would like to highlight the importance of rigorously annotating experimental structures and assigning their functional states. Finally, I would also like to highlight the power of molecular dynamics simulations to not only rigorously annotate experimental structures but also to provide atomistic details to explain experimental results. 

Spänningsstyrda natriumkanaler (Nav-kanaler) är viktiga för att leda nervimpulser i exciterbara celler. Således är denna kanal involverad i flera neurologiska och muskulära störningar. Att förstå deras mekanism är avgörande för att utforma läkemedel som har denna kanal som mål. Nav-kanaler är tetramera membranproteiner som selektivt transporterar natriumjoner över membranet. De reglerar jonflödet genom att växla mellan tre huvudsakliga funktionstillstånd - vilotillstånd, öppet tillstånd och inaktiverat tillstånd. Strukturbiologiska tekniker har funnit strukturer av Nav-kanaler i flera funktionella tillstånd, varav de flesta strukturerna fångas i ett inaktiverat tillstånd. Trots att det är utmanande att experimentellt komma fram till det öppna tillståndet på grund av dess kortlivade natur har flera strukturer av bakteriella och eukaryota Nav-kanaler fångats i det förmodade öppna tillståndet. Idag finns dessvärre ingen rigorös funktionell annotering av dessa strukturer i det öppna tillståndet.Molekylärdynamiska simuleringar visade att i de experimentellt lösta bakteriella Nav-kanaler som fångats i det förmodade öppna tillståndet, var poren dehydrerad och hade en hög fri energibarriär för passiv jon-/läkemedels transport, vilket tyder på att dessa strukturer inte motsvarar ett funktionellt öppet tillstånd. Helixarna som omringar poren i dessa protein är vanligtvis 𝛼-helix-karaktär, men sekvens-/strukturkonserveringsanalys i andra medlemmar av jonkanalsfamiljen visade att ibland kan 𝜋-helixar förekomma runt poren. Genom att introducera 𝜋-helixar i mitten av dessa pornära helixar hydratiserades poren och förminskade drastiskt den fria energi barriären för jon-/läkemedels genomträngning. 𝜋-helixarna kan också vara relevanta för poröppning eftersom de dehydrerar de perifera kaviteterna och minskar interaktionerna mellan de hydrofoba pornära regionerna och därmed tillåter öppning av den hydrofoba poren. Dessutom identifierades också en oordnad region i den C-terminala domänen som är känd för att vara relevant för poröppning.

Effekten av 𝜋-helixar på läkemedelstillgång och bindning till natriumkanaler studerades också. Resultatet var att 𝜋-helixar i den bakteriella Nav-kanalen blockerade laterala öppningar mot porregionen oberoende av pordiametern, vilket hämmade tillgången genom dessa laterala öppningar. Vidare undersöktes läkemedels bindning såväl som läkemedelsmolekylen Lidocains bindande till olika funktionella tillstånd, vilket visade att läkemedlet binder i olika orienteringar och positioner i olika funktionella tillstånd. Detta innebär att det kan finnas en förändring i den bindande affiniteten hos lidocain när kanalen växlar mellan olika funktionella tillstånd. Läkemedelsmolekylernas bindningsregion undersöktes också, vartill vägen för Cannabidiolbinding i natriumkanaler och effekten av Cannabidiol på membranets egenskaper kunde utforskas. Våra beräkningsresultat kompletterades med experimentella resultat. MD simuleringar tyder på att cannabis inte påverkade membranets styvhet och orsakade en ordning av membranets metylener, vilket är överensstämmer utmärkt med NMR-resultaten. Mutationsexperiment visar att cannabidiol blockerar porerna genom att interagera med en Fenylalanin-aminosyra som stämmer väl överens med våra docknings resultat. Adiabatically Biased Molekylärdynamiska simuleringar utfördes för att bekräfta vägen för CBD att nå porerna är genom dem laterala öppningarna i jonkanalen.

Idén om att 𝜋-helixar kan vara relevanta för poröppning generaliserades därefter till eukaryota Nav-kanaler. Eukaryota kanaler är heterotetrameriska, so poromringande helixar från olika homomerer kan påverka kanalens funktion på olika sätt. Genom MD simuleringar kunde det fastställas att antalet 𝜋-helixar i porregionen ökade inte bara hydreringen genom poren och jonkonduktants, men också förminskade barrirären för passiv jontransport. I synnerhet var 𝜋-helixar i homomer I och IV i det förstorade portillståndet viktiga för ett funktionellt öppet tillstånd.Genom att sammanställa ovanstående resultat demonstrerades de bakteriella experimentella strukturerna som ursprungligen föreslogs representera öppna tillstånd i stället kunde motsvara inaktiverade tillstånd. I eukaryoter motsvarar den experimentella strukturen som initialt föreslagits representera det öppna tillståndet ett öppet tillstånd under konduktans. Därför föreslås i detta arbete att en 𝜋 till 𝛼 helix övergång och vice versa kan vara relevant för reglering av poröppning i Nav-kanaler. Dessa resultat betonar vikten av att noggrant klassificera experimentella strukturer och tilldela deras funktionella tillstånd. Slutligen påvisades även kraften hos MD simuleringar, som kunde användas inte bara för att rigoröst klassificera experimentella strukturer utan också för att tillhandahålla atomistiska detaljer för att förklara experimentella resultat.

Voltage-gated sodium channels are heterotetrameric sodiumselectiveion channels that play a central role in electrical signaling in excitablecells. With recent advances in structural biology, structures of eukaryoticsodium channels have been captured in several distinct conformationscorresponding to different functional states. The secondary structureof the pore lining S6 helices of subunits DI, DII, and DIV has beencaptured with both short & pi;-helix stretches and in fully & alpha;-helicalconformations. The relevance of these secondary structure elementsfor pore gating is not yet understood. Here, we propose that a & pi;-helixin at least DI-S6, DIII-S6, and DIV-S6 results in a fully conductivestate. On the other hand, the absence of & pi;-helix in either DI-S6or DIV-S6 yields a subconductance state, and its absence from bothDI-S6 and DIV-S6 yields a nonconducting state. This work highlightsthe impact of the presence of a & pi;-helix in the different S6helices of an expanded pore on pore conductance, thus opening newdoors toward reconstructing the entire conformational landscape alongthe functional cycle of Nav Channels and paving the way to the design of state-dependent modulators.

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 pi-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 pi-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 pi-helix transition in the opening of NavAb, enabling new proposals for the structural annotation and drug modulation mechanisms in this important sodium channel model. We propose a new conformational cycle for NavAb wherein an alpha- to pi-helix transition in S6 and disordering of the neck region of the C-terminal domain is important for pore opening.

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 cc-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 p-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.

The K(V)7.4 and K(V)7.5 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 K(V)7.4 and K(V)7.5 channel function are of interest. Here, we study the effect of polyunsaturated fatty acids (PUFAs) on human K(V)7.4 and KV7.5 channels expressed in Xenopus oocytes. We report that PUFAs facilitate activation of hK(V)7.5 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 V-50 shifts. In contrast, PUFAs inhibit activation of hK(V)7.4 by shifting V-50 toward more positive voltages. No effect on V-50 of hK(V)7.4 is observed by an uncharged or a positively charged PUFA analogue. Thus, the hK(V)7.5 channel's response to PUFAs is analogous to the one previously observed in hK(V)7.1-7.3 channels, whereas the hK(V)7.4 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 hK(V)7.4 by PUFAs.

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 gramicidinbased 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 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.