Gating and modulation mechanism of voltage gated sodium channels

QC 2023-05-08

Abstract

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. 

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