Ion conduction and conformational flexibility of a bacterial voltage-gated sodium channel

Abstract

Voltage-gated Na + channels play an essential role in electrical signaling in the nervous system and are key pharmacological targets for a range of disorders. The recent solution of X-ray structures for the bacterial channel Na v Ab has provided an opportunity to study functional mechanisms at the atomic level. This channel's selectivity filter exhibits an EEEE ring sequence, characteristic of mammalian Ca 2+ , not Na + , channels. This raises the fundamentally important question: just what makes a Na + channel conduct Na + ions? Here we explore ion permeation on multimicrosecond timescales using the purpose-built Anton supercomputer. We isolate the likely protonation states of the EEEE ring and observe a striking flexibility of the filter that demonstrates the necessity for extended simulations to study conduction in this channel. We construct free energy maps to reveal complex multi-ion conduction via knock-on and "pass-by" mechanisms, involving concerted ion and glutamate side chain movements. Simulations in mixed ionic solutions reveal relative energetics for Na + , K + , and Ca 2+ within the pore that are consistent with the modest selectivity seen experimentally. We have observed conformational changes in the pore domain leading to asymmetrical collapses of the activation gate, similar to proposed inactivated structures of Na v Ab, with helix bending involving conserved residues that are critical for slow inactivation. These structural changes are shown to regulate access to fenestrations suggested to be pathways for lipophilic drugs and provide deeper insight into the molecular mechanisms connecting drug activity and slow inactivation.

A structural, functional, and computational analysis suggests pore flexibility as the base for the poor selectivity of CNG channels

“…The rotameric change in conformation of the Glu66 side chains herein observed is reminiscent of the mobility recently reported for Na + bacterial channels, where the conformational isomerization of a ring of four glutamate side chains lining the selectivity filter is coupled to ionic coordination (24,25,48). We propose that this flexibility, together with the number of equivalent and contiguous ion binding sites in the filter (26,34,49), underscores the poor ionic selectivity of CNG channels and reveals a conduction mode that differs substantially from that of classical K + channels, which are highly selective and have a fairly rigid molecular structure.…”

“…We demonstrate that the extracellular entrance of the selectivity filter and the filter itself exhibit a dynamic structure capable of structural rearrangements, which can be partially captured by X-ray crystallography and are best visualized and understood through MD simulations. Our results indicate that the pore of CNG channels is highly flexible, with a liquidlike energy landscape (24,25). This flexibility underlies the poor selectivity of CNG channels and their strong coupling between gating and permeation.…”

Voltage-Gated Sodium Channels

“…Similar conclusions were reached from long scale MD simulations (Boiteux et al, 2014), suggesting an essentially barrierless (~1.5 kcal/mol maximum barrier to conduction) multi ion 'knock-on' or 'pass-by' mechanism is more likely in the fully charged state, promoted by repulsive interactions between charged side-chains. The presence of one protonated glutamate, similarly, leads to a reduced charged density at the mouth of the selectivity filter.…”

“…Restriction of the selectivity filter, in a singly protonated system, seemingly adjusts the cavity shape and the orientation of S6 (Boiteux et al, 2014). The observed bent conformation in S6, on the level of P200-T206, is parallel to that of an inactivated structure suggested by Payandah et al (Payandeh et al 2012).…”

“…Subsequently, Boiteux and coworkers identified a double potential well at S HFS in PMF profiles, corresponding to available conformers of Glu177, reducing the barrier between selectivity filter sites (Boiteux, Vorobyov, & Allen, 2014). An average occupancy of 2.3 ions is reported in the selectivity filter, lending further evidence to a three-ion conduction mechanism in which the selectivity filter switches between one, two and three-ion configurations facilitated by the isomerization of Glu177.…”