Voltage-gated sodium channel (Na_V) protein dissection creates a set of functional pore-only proteins

Abstract

Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltagegated sodium channel (Na V Sp1) PD forms a stand-alone, ion selective pore (Na V Sp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the Na V Sp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel Na V Bh1 (NaChBac), creates a calcium-selective pore-only channel, Ca V Sp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile Na V s: one from the hydrocarbon degrader Alcanivorax borkumensis (Na V Ab1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (Na V Ae1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.V oltage-gated sodium channels (Na V s) are large polytopic membrane proteins involved in action potential generation in excitable cells and belong to an ion channel superfamily that includes voltage-gated calcium channels (Ca V s), voltage-gated potassium channels (K V s), and transient receptor potential (TRP) channels (1, 2). Within the voltage-gated ion channel (VGIC) superfamily, Na V s and Ca V s are close relatives (1-3) that share a topology of 24 transmembrane segments organized in four homologous six-transmembrane repeats. These two families are also thought to share some common structure in the ion selectivity filter despite having markedly different ion permeation properties (4). Both are central to human neuromuscular, cardiovascular, and neural physiology. Consequently, they are targets for a host of pharmaceuticals used to treat a diverse set of disorders and remain active targets for drug development (5-7). Recently, single subunit, six-transmembrane segment Na V s have been identified in a large number of bacteria from diverse environments (8, 9). These channels show clear similarities to eukaryotic Na V s and Ca V s (2, 9, 10), suggesting that the prokaryotic channels may have been ancestors of the more complex vertebrate channels.Despite the central importance of Na V s, nothing is known about the high-resolution structure o...

hERG S4-S5 linker acts as a voltage-dependent ligand that binds to the activation gate and locks it in a closed state

“…This ligand/receptor model implies that when S4-S5 L is absent, the hERG activation gate should be in the open state, independent of the potential, as observed in one potassium 22 and one sodium 23 bacterial channels. In other words, this gate is intrinsically more stable in the open state.…”

Molecular basis of ion permeability in a voltage‐gated sodium channel

“…Functional studies on wild‐type and mutant prokaryotic channels (Ren et al , 2001; Yue et al , 2002; McCusker et al , 2011; Shaya et al , 2011; Tang et al , 2014) have suggested which regions and residues may be important for ion binding, selectivity and translocation within the SF. However, the detailed locations and environments surrounding the sodium ions within the channels have not been discernable from the crystal structures published to date.…”

Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening

“…The PD is formed by the last two transmembrane segments (S5, S6) from four α-subunits and undergoes a mainly voltageindependent conformational change during which the intracellular ends of the S6 segments bend, opening the ionic pore (4,5). Interestingly, the PD and VSD can exist in pore-only (6) and voltage sensor-only proteins, respectively, where they function independently (7,8). Confining sensitivity to voltage, or to other stimuli, within a domain diversifies the ion channel properties that can be achieved by partnering different pore and sensor domains.…”