Mutagenesis of the NaChBac sodium channel discloses a functional role for a conserved S6 asparagine

Abstract

Asparagine is conserved in the S6 transmembrane segments of all voltage-gated sodium, calcium, and TRP channels identified to date. A broad spectrum of channelopathies including cardiac arrhythmias, epilepsy, muscle diseases, and pain disorders is associated with its mutation. To investigate its effects on sodium channel functional properties, we mutated the simple prokaryotic sodium channel NaChBac. Electrophysiological characterization of the N225D mutant reveals that this conservative substitution shifts the voltage-dependence of inactivation by 25 mV to more hyperpolarized potentials. The mutant also displays greater thermostability, as determined by synchrotron radiation circular dichroism spectroscopy studies of purified channels. Based on our analyses of high-resolution structures of NaChBac homologues, we suggest that the side-chain amine group of asparagine 225 forms one or more hydrogen bonds with different channel elements and that these interactions are important for normal channel function. The N225D mutation eliminates these hydrogen bonds and the structural consequences involve an enhanced channel inactivation.

Batrachotoxin acts as a stent to hold open homotetrameric prokaryotic voltage-gated sodium channels

“…Structures of eukaryotic Nav channels have recently become available (Shen et al, 2017, 2018; Yan et al, 2017), but it is unknown whether these represent functionally relevant Nav channel states. Nonetheless, the tractability of NavBac channels to coupled structural and functional study is clear (Payandeh et al, 2012; Shaya et al, 2014; Payandeh and Minor, 2015; Ahern et al, 2016; Arrigoni et al, 2016; O’Reilly et al, 2017; Sula et al, 2017; Chatterjee et al, 2018) and thus underlines the importance of prokaryotic Nav channels in the overall understanding of sodium channel function. Here, we have explored BTX actions in NaChBac and NavSp1, prokaryotic Nav channels for which much relevant structural information is available.…”

A consistent picture of TRPV1 activation emerges from molecular simulations and experiments

“…7A). In NaChBac, mutagenesis of this residue into several other amino acids yielded functional channels only in the case of N225D mutant; and even in that case gating of N225D was drastically affected (61). Furthermore, the structures of Nav and N676 I672 L673 L674 L678…”

Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4–S5 linker in activation and slow inactivation

“…Unlike the mutations at T140, a complementary mutation N225D in the S6 helix was found not to affect channel activation but profoundly shift the steady-state inactivation to the left ( 29 ). This, combined with the results in the current study, supports the notion that the S4–S5 linker initiates the conformation transition from the resting to the activation state by creating spaces for S6 kinking movement, whereas slow inactivation involves subsequent S6 helix rotation to orient N225 to face the S4–S5 linker to stabilize activation-coupled inactivation.…”

“…Except for its lack of a fast inactivation state, NaChBac shares many gating features with eukaryotic channels. It is ideally suited for studying Na V channel function because of its easy availability in large quantity and high purity from heterologous expressions in bacteria and mammalian cells (24)(25)(26)(27)(28)(29). It also has favorable gating kinetics for accurate electrorheology investigation into the transitions among different functional states (24).…”