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P2X receptor channels are trimeric ATP-activated ion channels expressed in neuronal and non-neuronal cells that are attractive therapeutic targets for human disorders. Seven subtypes of P2X receptor channels have been identified in mammals that can form both homomeric and heteromeric channels. P2X1–4 and P2X7 receptor channels are cation-selective, whereas P2X5 has been reported to have both cation and anion permeability. P2X receptor channel structures reveal that each subunit is comprised of two transmembrane helices, with both N-and C-termini on the intracellular side of the membrane and a large extracellular domain that contains the ATP binding sites at subunit interfaces. Recent structures of ATP-bound P2X receptors with the activation gate open reveal the unanticipated presence of a cytoplasmic cap over the central ion permeation pathway, leaving lateral fenestrations that may be largely buried within the membrane as potential pathways for ions to permeate the intracellular end of the pore. In the present study, we identify a critical residue within the intracellular lateral fenestrations that is readily accessible to thiol-reactive compounds from both sides of the membrane and where substitutions influence the relative permeability of the channel to cations and anions. Taken together, our results demonstrate that ions can enter or exit the internal pore through lateral fenestrations that play a critical role in determining the ion selectivity of P2X receptor channels.

Potassium (K + ) channels combine high conductance with high ion selectivity. To explain this efficiency, two molecular mechanisms have been proposed. The "direct knock-on" mechanism is defined by water-free K + permeation and formation of direct ion-ion contacts in the highly conserved selectivity filter (SF). The "soft knock-on" mechanism involves co-permeation of water and separation of K + by water molecules. With the aim to distinguish between these mechanisms, crystal structures of the KcsA channel with mutations in two SF residues-G77 and T75-were published, where the arrangements of K + ions and water display canonical soft knock-on configurations. These data were interpreted as evidence of the soft knock-on mechanism in wild-type channels. Here, we test this interpretation using molecular dynamics simulations of KcsA and its mutants. We show that while a strictly water-free direct knock-on permeation is observed in the wild type, conformational changes induced by these mutations lead to distinct ion permeation mechanisms, characterized by co-permeation of K + and water. These mechanisms are characterized by reduced conductance and impaired potassium selectivity, supporting the importance of full dehydration of potassium ions for the hallmark high conductance and selectivity of K + channels. In general, we present a case where mutations introduced at the critical points of the permeation pathway in an ion channel drastically change its permeation mechanism in a nonintuitive manner., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

A novel artificial cation channel was developed by rebuilding the ion permeation pathway of the natural channel protein (TRPA1) in a synthetic system. This tubular molecule can effectively embed into lipid bilayers and form transmembrane channels, thereby mediating cation transport. Furthermore, due to its carboxyl-modified ion permeation pathway, the transport activity of this artificial channel can be modulated by the pH of the buffer solution., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Intracellular divalent cations control the molecular function of transmembrane protein 16 (TMEM16) family members. Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) in this family are activated by intracellular Ca2+ in the low µM range. In addition, intracellular Ca2+ or Co2+ at mM concentrations have been shown to further potentiate the saturated Ca2+-activated current of TMEM16A. In this study, we found that all alkaline earth divalent cations in mM concentrations can generate similar potentiation effects in TMEM16A when applied intracellularly, and that manipulations thought to deplete membrane phospholipids weaken the effect. In comparison, mM concentrations of divalent cations minimally potentiate the current of TMEM16F but significantly change its cation/anion selectivity. We suggest that divalent cations may increase local concentrations of permeant ions via a change in pore electrostatic potential, possibly acting through phospholipid head groups in or near the pore. Monovalent cations appear to exert a similar effect, although with a much lower affinity. Our findings resolve controversies regarding the ion selectivity of TMEM16 proteins. The physiological role of this mechanism, however, remains elusive because of the nearly constant high cation concentrations in cytosols.

Protein dynamics are essential to function. One example of this is the various gating mechanisms within ion channels, which are transmembrane proteins that act as gateways into the cell. Typical ion channels switch between an open and closed state via a conformational transition which is often triggered by an external stimulus, such as ligand binding or pH and voltage differences. The atomic resolution structure of a potassium-selective ion channel named NaK2K has allowed us to observe that a hydro-phobic residue at the bottom of the selectivity filter, Phe92, appears in dual conformations. One of the two conformations of Phe92 restricts the diameter of the exit pore around the selectivity filter, limiting ion flow through the channel, while the other conformation of Phe92 provides a larger-diameter exit pore from the selectivity filter. Thus, it can be concluded that Phe92 acts as a hydro-phobic gate, regulating the flow of ions through the selectivity filter.

Natural redox-regulated channel proteins often utilize disulfide bonds as redox sensors for adaptive regulation of channel conformations in response to diverse physiological environments. In this study, we developed novel synthetic ion channels capable of reversibly switching their ion-transport capabilities by incorporating multiple disulfide bonds into artificial systems. X-ray structural analysis and electrophysiological experiments demonstrated that these disulfide-bridged molecules possess well-defined tubular cavities and can be efficiently inserted into lipid bilayers to form artificial ion channels. More importantly, the disulfide bonds in these molecules serve as redox-tunable switches to regulate the formation and disruption of ion-permeation pathways, thereby achieving a transition in the transmembrane transport process between the ON and OFF states., (© 2024 Wiley-VCH GmbH.)

Protein dynamics are essential to function. One example of this is the various gating mechanisms within ion channels, which are transmembrane proteins that act as gateways into the cell. Typical ion channels switch between an open and closed state via a conformational transition which is often triggered by an external stimulus, such as ligand binding or pH and voltage differences. The atomic resolution structure of a potassium-selective ion channel named NaK2K has allowed us to observe that a hydrophobic residue at the bottom of the selectivity filter, Phe92, appears in dual conformations. One of the two conformations of Phe92 restricts the diameter of the exit pore around the selectivity filter, limiting ion flow through the channel, while the other conformation of Phe92 provides a larger-diameter exit pore from the selectivity filter. Thus, it can be concluded that Phe92 acts as a hydrophobic gate, regulating the flow of ions through the selectivity filter.

Summary: Recently identified proton-activated chloride channel (PAC) contains two transmembrane helices (S1–S2) and is involved in lysosome function, hypoxia adaption, stroke, and carcinogenesis. Although a PAC structure was recently resolved, its gating and activation mechanisms remained largely unknown. By the two-electrode voltage clamp electrophysiology in Xenopus oocytes, we found that the hydrophobicity of site 304 at fenestrations, but not that of neighbor sites, is important for maintaining PAC at a closed state at pH 7.5. When activated at acidic pH, PAC activity significantly increased with the hydrophilicity of site 307 within S2, but not with that of neighbor sites, suggesting that 307 acts as an activation gate. We identified six conserved protonatable residues critical for proton-induced activation, consistent with structural studies. Our study depicted a scheme in which proton binding induces conformational changes from the W304-controlled closed state at fenestrations to an activated state controlled by activation gate I307 in helix S2.

Ion transport molecules are involved in many physiological and pathological processes and are considered potential targets for cancer treatment. In the large family of ion transport molecules, potassium (K) ion channels, as surface-expressed proteins, show the highest variability and most frequent expression changes in many tumor types. The key to exploring the permeation of K+ through potassium channels lies in the conserved sequence TVGYG, which is common in the selectivity filter (SF) region of all potassium channels. We found that the K+ flux significantly increased with the help of a specific frequency terahertz electromagnetic wave (51.87 THz) in the KcsA channel using a molecular dynamics combined model through the combined simulation of the constant electric field method and ion imbalance method. This frequency has the strongest absorption peak in the infrared spectrum of -C=O groups in the SF region. With the applied electric field of 51.87 THz, the Y78 residue at the S1 site of the SF has a smaller vibration amplitude and a more stable structure, which enables the K+ to bind closely with the carbonyl oxygen atoms in the SF and realize ion conduction in a more efficient direct Coulomb knock-on.

P2X receptor channels are trimeric ATP-activated ion channels expressed in neuronal and non-neuronal cells that are attractive therapeutic targets for human disorders. Seven subtypes of P2X receptor channels have been identified in mammals that can form both homomeric and heteromeric channels. P2X1-4 and P2X7 receptor channels are cation-selective, whereas P2X5 has been reported to have both cation and anion permeability. P2X receptor channel structures reveal that each subunit is comprised of two transmembrane helices, with both N-and C-termini on the intracellular side of the membrane and a large extracellular domain that contains the ATP binding sites at subunit interfaces. Recent structures of ATP-bound P2X receptors with the activation gate open reveal the unanticipated presence of a cytoplasmic cap over the central ion permeation pathway, leaving lateral fenestrations that may be largely buried within the membrane as potential pathways for ions to permeate the intracellular end of the pore. In the present study, we identify a critical residue within the intracellular lateral fenestrations that is readily accessible to thiol-reactive compounds from both sides of the membrane and where substitutions influence the relative permeability of the channel to cations and anions. Taken together, our results demonstrate that ions can enter or exit the internal pore through lateral fenestrations that play a critical role in determining the ion selectivity of P2X receptor channels., Competing Interests: ST, KH, ML No competing interests declared, KS Senior editor, eLife

Whether ion channel gating is independent of ion permeation has been an enduring, unresolved question. Here, applying single channel recording to the archetypal muscle nicotinic receptor, we unmask coupling between channel gating and ion permeation by structural perturbation of a conserved intramembrane salt bridge. A charge-neutralizing mutation suppresses channel gating, reduces unitary current amplitude, and increases fluctuations of the open channel current. Power spectra of the current fluctuations exhibit low- and high-frequency Lorentzian components, which increase in charge-neutralized mutant receptors. After aligning channel openings and closings at the time of transition, the average unitary current exhibits asymmetric relaxations just after channel opening and before channel closing. A theory in which structural motions contribute jointly to channel gating and ion conduction describes both the power spectrum and the current relaxations. Coupling manifests as a transient increase in the open channel current upon channel opening and a decrease upon channel closing.