Putting Together the Puzzle of Ion Transfer in Single-Digit Carbon Nanotubes: Ab Initio Meets Mean-Field

Nature employs channel proteins to selectively pass water across cell membranes, which inspires search for bio-mimetic analogues. Carbon nanotube porins (CNTPs) are intriguing mimics of water channels, yet ion transport in CNTPs still poses questions. As alternative to continuum models, here we present a molecular mean-field model, computing ab initio all required thermodynamic quantities for KCl salt and H+ and OH- ions present in water. Starting from water transfer, the model considers transfer of free ions, along with ion-pair formation to address ion-ion interactions. High affinity to hydroxide, suggested by experiments and making it dominant charge carrier in CNTP, is revealed as an exceptionally favorable transfer of KOH pairs. Nevertheless, free ions, coexisting with less mobile ion-pairs, apparently control ion transport. The model explains well the observed effects of salt concentration and pH on conductivity, transport numbers, anion permeation and its activation energies, and current rectification. The proposed approach is extendable to other sub-nanochannels and help design novel osmotic materials and devices.

Nonselective cation permeation in an AMPA-type glutamate receptor

Fast excitatory synaptic transmission in the central nervous system relies on the AMPA-type glutamate receptor (AMPAR). This receptor incorporates a nonselective cation channel, which is opened by the binding of glutamate. Although the open pore structure has recently became available from cryo-electron microscopy (Cryo-EM), the molecular mechanisms governing cation permeability in AMPA receptors are not understood. Here, we combined microsecond molecular dynamic (MD) simulations on a putative open-state structure of GluA2 with electrophysiology on cloned channels to elucidate ion permeation mechanisms. Na+, K+, and Cs+ permeated at physiological rates, consistent with a structure that represents a true open state. A single major ion binding site for Na+ and K+ in the pore represents the simplest selectivity filter (SF) structure for any tetrameric cation channel of known structure. The minimal SF comprised only Q586 and Q587, and other residues on the cytoplasmic side formed a water-filled cavity with a cone shape that lacked major interactions with ions. We observed that Cl− readily enters the upper pore, explaining anion permeation in the RNA-edited (Q586R) form of GluA2. A permissive architecture of the SF accommodated different alkali metals in distinct solvation states to allow rapid, nonselective cation permeation and copermeation by water. Simulations suggested Cs+ uses two equally populated ion binding sites in the filter, and we confirmed with electrophysiology of GluA2 that Cs+ is slightly more permeant than Na+, consistent with serial binding sites preferentially driving selectivity.

Mutations in an intracellular vestibule that bifurcates from the pore of ClC-1 chloride ion channels affect anion permeation

The ubiquitous CLC protein superfamily consists of channels, that permit passive diffusion of Cl ions across biological membranes, and pumps, that can actively transport Cl ions against their electrochemical gradient; yet, puzzlingly, both types share a strongly conserved Cl ion transport pathway comprised of three consecutive binding sites. This raises the question; how does the same pathway support passive diffusion in CLC channels and active transport in CLC pumps? Based on high-resolution structural data current theories suggest that subtle structural differences in the conserved pathway allow CLC channels to ‘leak’ Cl ions. A recent cryo-electron microscopy structure of the human ClC-1 channel does not show occupancy of the central Cl ion binding site but reveals a wide intracellular vestibule that bifurcates from the conserved pathway in this region. Here we show that replacing residues that line the ClC-1 intracellular vestibule with the corresponding residues of CLC pumps resulted in interactions between permeating anions at neighbouring binding sites and altered anion selectivity. Removing the side chain of a strictly conserved tyrosine residue, that coordinates Cl ion at the central binding site of CLC pumps, removed multi-ion behaviour in ClC-1 mutants. In contrast, removing the side chain of a highly conserved glutamate residue that transiently occupies Cl ion binding sites, as part of the transport mechanism of CLC pumps and the mechanism that opens and closes CLC channels, only partially removed multi-ion behaviour in ClC-1 mutants. Our findings show that structural differences between CLC channels and pumps, outside of the conserved Cl ion transport pathway, fundamentally affect anion permeation in ClC-1 channels.SummarySome CLC proteins are passive Cl- channels while others are active Cl- pumps but, paradoxically, both share a conserved, canonical, Cl- permeation pathway. Here Bennetts, Morton and Parker show that ‘pump-like’ mutations in a poorly conserved region, located remotely from the canonical pathway, affect anion permeation in human ClC-1 channels.

Combinatorial expression of claudins in the proximal renal tubule and its functional consequences

The proximal renal tubule (PT) is characterized by a highly conductive paracellular pathway, which contributes to a significant amount of solute and water reabsorption by the kidney. Claudins are tight junction proteins that, in part, determine the paracellular permeability of epithelia. In the present study, we determined the expression pattern of the major PT claudins. We found that claudin-2 and claudin-10 are coexpressed throughout the PT, whereas claudin-3 is coexpressed with claudin-2 predominantly in the proximal straight tubule. Additionally, claudin-2 and claudin-3 are expressed separately within mutually exclusive populations of descending thin limbs. We developed a novel double-inducible Madin-Darby canine kidney I cell model to characterize in vitro the functional effect of coexpression of PT claudins. In keeping with previous studies, we found that claudin-2 alone primarily increased cation (Na+ and Ca2+) permeability, whereas claudin-10a alone increased anion (Cl−) permeability. Coexpression of claudin-2 and claudin-10a together led to a weak physical interaction between the isoforms and the formation of a monolayer with high conductance but neutral charge selectivity. Claudin-3 expression had a negligible effect on all measures of cell permeability, whether expressed alone or together with claudin-2. In cells coexpressing a claudin-2 mutant, S68C, together with claudin-10a, inhibition of cation permeability through the claudin-2 pore with a thiol-reactive pore blocker did not block anion permeation through claudin-10a. We conclude that claudin-2 and claudin-10a form independent paracellular cation- and anion-selective channels that function in parallel.

Substrate transport and anion permeation proceed through distinct pathways in glutamate transporters

Advances in structure-function analyses and computational biology have enabled a deeper understanding of how excitatory amino acid transporters (EAATs) mediate chloride permeation and substrate transport. However, the mechanism of structural coupling between these functions remains to be established. Using a combination of molecular modeling, substituted cysteine accessibility, electrophysiology and glutamate uptake assays, we identified a chloride-channeling conformer, iChS, transiently accessible as EAAT1 reconfigures from substrate/ion-loaded into a substrate-releasing conformer. Opening of the anion permeation path in this iChS is controlled by the elevator-like movement of the substrate-binding core, along with its wall that simultaneously lines the anion permeation path (global); and repacking of a cluster of hydrophobic residues near the extracellular vestibule (local). Moreover, our results demonstrate that stabilization of iChS by chemical modifications favors anion channeling at the expense of substrate transport, suggesting a mutually exclusive regulation mediated by the movement of the flexible wall lining the two regions.

Spatial positioning of CFTR’s pore-lining residues affirms an asymmetrical contribution of transmembrane segments to the anion permeation pathway

The structural composition of CFTR’s anion permeation pathway has been proposed to consist of a short narrow region, flanked by two wide inner and outer vestibules, based on systematic cysteine scanning studies using thiol-reactive probes of various sizes. Although these studies identified several of the transmembrane segments (TMs) as pore lining, the exact spatial relationship between pore-lining elements remains under debate. Here, we introduce cysteine pairs in several key pore-lining positions in TM1, 6, and 12 and use Cd2+ as a probe to gauge the spatial relationship of these residues within the pore. We find that inhibition of single cysteine CFTR mutants, such as 102C in TM1 or 341C in TM6, by intracellular Cd2+ is readily reversible upon removal of the metal ion. However, the inhibitory effect of Cd2+ on the double mutant 102C/341C requires the chelating agent dithiothreitol (DTT) for rapid reversal, indicating that 102C and 341C are close enough to the internal edge of the narrow region to coordinate one Cd2+ ion between them. We observe similar effects of extracellular Cd2+ on TM1/TM6 cysteine pairs 106C/337C, 107C/337C, and 107C/338C, corroborating the idea that these paired residues are physically close to each other at the external edge of the narrow region. Although these data paint a picture of relatively symmetrical contributions to CFTR’s pore by TM1 and TM6, introducing cysteine pairs between TM6 and TM12 (348C/1141C, 348C/1144C, and 348C/1145C) or between TM1 and TM12 (95C/1141C) yields results that contest the long-held principle of twofold pseudo-symmetry in the assembly of ABC transporters’ TMs. Collectively, these findings not only advance our current understanding of the architecture of CFTR’s pore, but could serve as a guide for refining computational models of CFTR by imposing physical constraints among pore-lining residues.