Mutational analysis of state-dependent contacts in the pore module of eukaryotic sodium channels
Introduction
Sodium channels play key roles in physiology of excitable cells. The alpha-subunit of the channels folds from a single polypeptide chain that forms four homologous repeats (I, II, III, and IV) arranged clockwise when viewed from the extracellular side [11]. Each repeat has six membrane spanning helical segments (S1–S6). Segments S1–S4 constitute the voltage-sensing domains, which are connected to the pore domain through the linker-helices (L45). The pore domain contains four outer helices S5, four pore-lining inner helices S6, and four membrane-reentrant P-loops. Upon membrane depolarization segments S4 move in the extracellular direction, initiating opening of the activation gate formed by residues at the cytoplasmic parts of S6s. The ring of D, E, K and A residues provided by P-helices in repeats I-IV, respectively, forms the selectivity filter that divides the permeation pathway in two parts: the outer pore exposed to the extracellular space, and the inner pore, which upon the channel activation opens to the cytoplasm. The cytoplasmic linker between repeats III and IV contains the fast-inactivation latch (IFM motif), which blocks the ion permeation [29,33,35]. The channel recovers from the fast inactivation when the latch unbinds from its receptor. Long depolarization or trains of action potential over hundreds milliseconds cause slow inactivation, which involves conformational changes in both the outer and the inner pore [14,28].
The pore-lining inner helices contain exceptionally conserved asparagines, NXi20s, six positions downstream from the putative gating hinge residues GXi14.2 The exceptional conservation suggests important functional roles. Earlier we proposed that the conserved asparagines NXi20s stabilize the open channel and shape the open-pore geometry by forming state-dependent interdomain H-bonds with polar residues at the C-ends of the preceding-domain inner helices, typically in positions (X-1)i29 [25]. Available structures do not provide unambiguous view on the role of the conserved asparagines. Effects of substitutions of NXi20s have been explored in electrophysiological studies, e.g. Refs. [8,[19], [20], [21],30,31,36]. However, putative open-state H-bonding partners of NXi20s have not yet been studied in mutational and electrophysiological experiments.
Insect sodium channels contain polar residues in positions i29 of all the four inner helices. Therefore, these channels are convenient objects to explore how possible inter-repeat open-state H-bonds involving conserved asparagines could affect electrophysiological characteristics. Here we mutated the conserved asparagines, their putative open-state H-bonding partners in positions i29 and some other residues, and analyzed impact of the mutations on the channel activation, fast inactivation, and slow inactivation. Analysis of our data in view of available open- and closed-state structures supports the hypothesis that inter-repeat H-bonds involving the conserved asparagines Ni20s and residues in positions i29 contribute to stabilization of the open-state conformation.
Section snippets
Molecular biology
Fig. 1A shows positions in which alanine substitutions were generated in the mosquito (AaNav1-1) and cockroach (BgNav1-1a) sodium channels. Site-directed mutagenesis was performed by Polymerase Chain Reaction (PCR) using Phusion High-Fidelity DNA polymerase (NEB, Ipswich, MA). All mutagenesis results were confirmed by DNA sequencing. The AaNav1-1 and BgNav1-1a channels and their mutants were expressed in Xenopus laevis oocytes. The procedures for oocyte preparation and cRNA injection are
Site-directed mutagenesis
To explore roles of possible state-dependent inter-repeat H-bonds involving the conserved asparagines we used mutations N2i20A and S1i29A, which are available from our previous study of the mosquito sodium channel AaNav1-1 [10], and generated six new mutants of the channel: N1i20A, N4i29A, N3i20A, N2i29A, N4i20A and N3i29A. Regrettably, the AaNav1-1 mutants N3i20A and N4i20A did not generate currents. This may be due to either poorly expressed or non-functional mutants. Therefore, we used the
Concluding remarks
In this study we used insect sodium channels to test the hypotheses that conserved asparagines in positions i20 of the inner helices form open-state inter-repeat H-bonds with polar residues at the C-ends of preceding-repeat inner helices [25]. We mutated residues in positions i20 and i29 and explored electrophysiological characteristics of the mutants.
Asparagines Ni20 are exceptionally conserved in the S6 helices of voltage-gated sodium and calcium channels. A systematic analysis of alanine
Acknowledgements
This study was supported by grants from the National Institute of General Medical Sciences (GM057440), Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-04894), and Russian Foundation for Basic Research (17-04-00549). Computations were performed using the facilities of the Shared Hierarchical Academic Research Computing Network (SHARCNET, www.sharcnet.ca).
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