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The solute carrier family 12A (SLC12A) superfamily of membrane transporters modulates the movement of cations coupled with chloride across the membrane. In doing so, these cotransporters are involved in numerous aspects of human physiology: cell volume regulation, ion homeostasis, blood pressure regulation, and neurological action potential via intracellular chloride concentration modulation. Their physiological characterization has been largely studied; however, understanding the mechanics of their function and the relevance of structural domains or specific amino acids has been a pending task. In recent years, singleparticle cryogenic electron microscopy (cryo-EM) has been successfully applied to members of the SLC12A family including all K+:Cl- cotransporters (KCCs), Na+:K+:2Cl- cotransporter NKCC1, and recently Na+:Cl- cotransporter (NCC); revealing structural elements that play key roles in their function. The present review analyzes the data provided by these cryo-EM reports focusing on structural domains and specific amino acids involved in ion binding, domain interactions, and other important SCL12A structural elements. A comparison of cryo-EM data from NKCC1 and KCCs is presented in the light of the two recent NCC cryo-EM studies, to propose insight into structural elements that might also be found in NCC and are necessary for its proper function. In the final sections, the importance of key coordination residues for substrate specificity and their implication on various pathophysiological conditions and genetic disorders is reviewed, as this could provide the basis to correlate structural elements with the development of novel and selective treatments, as well as mechanistic insight into the function and regulation of cationcoupled chloride cotransporters (CCCs). [ABSTRACT FROM AUTHOR]

The plasma-membrane homeostasis Na+/Ca2+ exchangers (NCXs) mediate Ca2+ extrusion/entry to dynamically shape Ca2+ signaling/in biological systems ranging from bacteria to humans. The NCX gene orthologs, isoforms, and their splice variants are expressed in a tissue-specific manner and exhibit nearly 104-fold differences in the transport rates and regulatory specificities to match the cell-specific requirements. Selective pharmacological targeting of NCX variants could benefit many clinical applications, although this intervention remains challenging, mainly because a full-size structure of eukaryotic NCX is unavailable. The crystal structure of the archaeal NCX_Mj, in conjunction with biophysical, computational, and functional analyses, provided a breakthrough in resolving the ion transport mechanisms. However, NCX_Mj (whose size is nearly three times smaller than that of mammalian NCXs) cannot serve as a structure-dynamic model for imitating high transport rates and regulatory modules possessed by eukaryotic NCXs. The crystal structures of isolated regulatory domains (obtained from eukaryotic NCXs) and their biophysical analyses by SAXS, NMR, FRET, and HDX-MS approaches revealed structure-based variances of regulatory modules. Despite these achievements, it remains unclear how multi-domain interactions can decode and integrate diverse allosteric signals, thereby yielding distinct regulatory outcomes in a given ortholog/isoform/splice variant. This article summarizes the relevant issues from the perspective of future developments. [ABSTRACT FROM AUTHOR]

EAG1 channels belong to the KCNH family of voltage gated potassium channels. They are expressed in several brain regions and increased expression is linked to certain cancer types. Recent cryo-EM structure determination finally revealed the structure of these channels in atomic detail, allowing computational investigations. In this study, we performed molecular dynamics simulations to investigate the ion binding sites and the dynamical behavior of the selectivity filter. Our simulations suggest that sites S2 and S4 form stable ion binding sites, while ions placed at sites S1 and S3 rapidly switched to sites S2 and S4. Further, ions tended to dissociate away from S0 within less than 20 ns, due to increased filter flexibility. This was followed by water influx from the extracellular side, leading to a widening of the filter in this region, and likely non-conductive filter configurations. Simulations with the inactivation-enhancing mutant Y464A or Na + ions lead to trapped water molecules behind the SF, suggesting that these simulations captured early conformational changes linked to C-type inactivation. [ABSTRACT FROM AUTHOR]

The plasma-membrane homeostasis Na + /Ca 2+ exchangers (NCXs) mediate Ca 2+ extrusion/entry to dynamically shape Ca 2+ signaling/in biological systems ranging from bacteria to humans. The NCX gene orthologs, isoforms, and their splice variants are expressed in a tissue-specific manner and exhibit nearly 10 4 -fold differences in the transport rates and regulatory specificities to match the cell-specific requirements. Selective pharmacological targeting of NCX variants could benefit many clinical applications, although this intervention remains challenging, mainly because a full-size structure of eukaryotic NCX is unavailable. The crystal structure of the archaeal NCX_Mj, in conjunction with biophysical, computational, and functional analyses, provided a breakthrough in resolving the ion transport mechanisms. However, NCX_Mj (whose size is nearly three times smaller than that of mammalian NCXs) cannot serve as a structure-dynamic model for imitating high transport rates and regulatory modules possessed by eukaryotic NCXs. The crystal structures of isolated regulatory domains (obtained from eukaryotic NCXs) and their biophysical analyses by SAXS, NMR, FRET, and HDX-MS approaches revealed structure-based variances of regulatory modules. Despite these achievements, it remains unclear how multi-domain interactions can decode and integrate diverse allosteric signals, thereby yielding distinct regulatory outcomes in a given ortholog/isoform/splice variant. This article summarizes the relevant issues from the perspective of future developments.

Abstract: Rapid and selective ion transport is essential for the generation and regulation of electrical signaling pathways in living organisms. In this study, we use molecular dynamics simulations and free energy calculations to investigate how the bacterial sodium channel NaVAb (Arcobacter butzleri) differentiates between Na+ and Ca2+ ions. Multiple nanosecond molecular dynamics simulations revealed distinct binding patterns for these two cations in the selectivity filter and suggested a high affinity calcium binding site formed by backbone atoms of residues Leu-176 and Thr-175 (SCEN) in the sodium channel selectivity filter. [Copyright &y& Elsevier]

The recent crystal structure of the prokaryotic inwardly rectifying potassium channel, KirBac1.1, revealed for the first time the structure of a K+ channel in the closed state plus the location of the activation gate. Comparison of the KirBac1.1 structure with other known ion channels reveals a number of common structural features. These common characteristics include the formation of the ion conduction pathway at the interface between adjacent subunits, non-fixed charges forming part of the ion pathway, electrostatic sinks drawing ions into the channel, helix dipoles, and hydrophobic gates that ultimately prevent ion movement. This review describes in detail common structural themes present in ion channels. [ABSTRACT FROM AUTHOR]

A family of fluorescent styryl dyes was synthesized to apply them as probes that monitor the ion-translocating activity of the Na,K-ATPase and the SR Ca-ATPase, similar to the widely used dye RH421. All dyes had the same chromophore but they differed in the length of the spacer between chromophore and polar head, an isothiocyanate group, and in the lengths of the two identical acyl chains, which form the tail of the dye molecules. A number of substrate-dependent partial reactions of both P-type ATPases affected the fluorescence intensity, and the magnitude of the fluorescence changes was used to characterize the usefulness of the dyes for further application. The experimental results indicate that electrochromy is the major mechanism of these dyes. While in the case of the Na,K-ATPase a single dye, 5QITC, showed larger fluorescence changes than all others, in the case of the SR Ca-ATPase all dyes tested were almost equal in their fluorescence responses. This prominent difference is interpreted as a hint that the position of the ion binding sites in both ion pumps may differ significantly despite their otherwise closely related structural features. Quench experiments with spin-labeled lipids in various positions of their fatty acids were used to gain information on the depth of the chromophore of the different dyes within the membrane dielectric, however, the spatial resolution was so poor that only qualitative information on the position of the chromophore in the lipid phase could be obtained. [ABSTRACT FROM AUTHOR]