Covalent linkage of bacterial voltage-gated sodium channels

Abstract

Background Bacterial sodium channels are important models for understanding ion permeation and selectivity. However, their homotetrameric structure limits their use as models for understanding the more complex eukaryotic voltage-gated sodium channels (which have a pseudo-heterotetrameric structure formed from an oligomer composed of four domains). To bridge this gap we attempted to synthesise oligomers made from four covalently linked bacterial sodium channel monomers and thus resembling their eukaryotic counterparts. Results Western blot analyses revealed NaChBac oligomers to be inherently unstable whereas intact expression of NavMs oligomers was possible. Immunodectection using confocal microscopy and electrophysiological characterisation of NavMs tetramers confirmed plasma membrane localisation and equivalent functionality with wild type NavMs channels when expressed in human embryonic kidney cells. Conclusion This study has generated new tools for the investigation of eukaryotic channels. The successful covalent linkage of four bacterial Nav channel monomers should permit the introduction of radial asymmetry into the structure of bacterial Nav channels and enable the known structures of these channels to be used to gain unique insights into structure-function relationships of their eukaryotic counterparts. Electronic supplementary material The online version of this article (10.1186/s13628-019-0049-5) contains supplementary material, which is available to authorized users.

Changes in Ion Selectivity Following the Asymmetrical Addition of Charge to the Selectivity Filter of Bacterial Sodium Channels

“…First, mixed populations of NaChBac monomers (differing in their amino acid composition and the Q f value of the SF) were co-transfected into CHO cells to generate hetero-tetrameric channels exhibiting radial asymmetry in the SFs. Second, we used a concatenated NavMs tetramer [ 38 ] to generate radial asymmetry in the SF by the targeted mutation of one of the four repeats.…”

“…Although the use of a mixed population of cDNAs encoding for NaChBac and its mutants suggested the value of Q f to be a major determining factor for Na + /Ca 2+ selectivity, the results are subject to the caveat that the whole-cell currents result from the cumulative current from an unknown but predictable range of different channel types. To address this complication, we attempted to generate a stable concatenation of NaChBac to enable the expression of a homogeneous population of NaChBac mutants; however, we have previously shown [ 38 ] the NaChBac oligomer to be unstable and not to remain intact in the plasma membrane. In contrast, an equivalent intact NavMs oligomer could be stably expressed in HEK293T cells [ 38 ] and thus enable the generation of a homogeneous population of bacterial channels, in which the Q f value of the SF can be altered in steps of 1 e .…”

“…To address this complication, we attempted to generate a stable concatenation of NaChBac to enable the expression of a homogeneous population of NaChBac mutants; however, we have previously shown [ 38 ] the NaChBac oligomer to be unstable and not to remain intact in the plasma membrane. In contrast, an equivalent intact NavMs oligomer could be stably expressed in HEK293T cells [ 38 ] and thus enable the generation of a homogeneous population of bacterial channels, in which the Q f value of the SF can be altered in steps of 1 e . The SF of eukaryotic CaVs is formed by a ring of glutamates (the EEEE locus) and a conserved aspartate residue in domain II (D2p51 [ 37 ]).…”

“…cDNA constructs encoding NaChBac (GenBank accession number BAB05220) and NavMs (GenBank accession number WP_011712479) bacterial sodium channels were synthesized by EPOCH Life Science ( ). NavMs concatemer was subcloned into pTRACER-CMV2 (Invitrogen) downstream of CMV promoter, as described previously [ 38 ].…”

“…The different behavior of prokaryotic and eukaryotic voltage-gated sodium and calcium channels highlights the importance of incorporating radial asymmetry in the SF of prokaryotic channels. In our previous work [ 38 ] we reported the creation of a concatenated bacterial NaV, in which four NavMs monomers were covalently linked to form a stable single polypeptide chain, resembling the general structure of a eukaryotic NaV. This allowed the targeted mutagenesis of individual domains introducing radial asymmetry in the bacterial channel with the aim to gain further insight on the role of Q f as a determinant of ion selectivity.…”