P2X receptor channels show threefold symmetry in ionic charge selectivity and unitary conductance.
Ontology highlight
ABSTRACT:
SUBMITTER: Browne LE
PROVIDER: S-EPMC3012030 | biostudies-literature | 2011 Jan
REPOSITORIES: biostudies-literature
Publications
P2X receptor channels show threefold symmetry in ionic charge selectivity and unitary conductance.
Browne Liam E LE Cao Lishuang L Broomhead Helen E HE Bragg Laricia L Wilkinson William J WJ North R Alan RA
Nature neuroscience 20101219 1
In the closed structure of the P2X cation channel, three α-helical transmembrane domains cross the membrane obliquely. In rat P2X2 receptors, these intersect at Thr(339). Replacing Thr(339) by lysine in one, two or three subunits progressively increased chloride permeability and reduced unitary conductance. This implies that the closed-open transition involves a symmetrical separation of the three subunits and that Thr(339) from each subunit contributes symmetrically to the open channel permeati ...[more]
Similar Datasets
Project description:K channels mediate the selective passage of K(+) across the plasma membrane by means of intimate interactions with ions at the pore selectivity filter located near the external face. Despite high conservation of the selectivity filter, the K(+) transport properties of different K channels vary widely, with the unitary conductance spanning a range of over two orders of magnitude. Mutation of Pro475, a residue located at the cytoplasmic entrance of the pore of the small-intermediate conductance K channel Shaker (Pro475Asp (P475D) or Pro475Gln (P475Q)), increases Shaker's reported ? 20-pS conductance by approximately six- and approximately threefold, respectively, without any detectable effect on its selectivity. These findings suggest that the structural determinants underlying the diversity of K channel conductance are distinct from the selectivity filter, making P475D and P475Q excellent probes to identify key determinants of the K channel unitary conductance. By measuring diffusion-limited unitary outward currents after unilateral addition of 2 M sucrose to the internal solution to increase its viscosity, we estimated a pore internal radius of capture of ? 0.82 Å for all three Shaker variants (wild type, P475D, and P475Q). This estimate is consistent with the internal entrance of the Kv1.2/2.1 structure if the effective radius of hydrated K(+) is set to ? 4 Å. Unilateral exposure to sucrose allowed us to estimate the internal and external access resistances together with that of the inner pore. We determined that Shaker resistance resides mainly in the inner cavity, whereas only ? 8% resides in the selectivity filter. To reduce the inner resistance, we introduced additional aspartate residues into the internal vestibule to favor ion occupancy. No aspartate addition raised the maximum unitary conductance, measured at saturating [K(+)], beyond that of P475D, suggesting an ? 200-pS conductance ceiling for Shaker. This value is approximately one third of the maximum conductance of the large conductance K (BK) channel (the K channel of highest conductance), reducing the energy gap between their K(+) transport rates to ? 1 kT. Thus, although Shaker's pore sustains ion translocation as the BK channel's does, higher energetic costs of ion stabilization or higher friction with the ion's rigid hydration cage in its narrower aqueous cavity may entail higher resistance.
| S-EPMC4516780 | biostudies-literature
Project description:The surface charge effect in controlling ionic conductance through a nanoporous alumina membrane is investigated for its application in a convenient detection method of unlabeled DNA. To this goal, surface modification with mixtures of neutral silanes and morpholinos (neutral analogues of DNA) was optimized to yield a strong effect on ionic conductance change upon DNA binding, which can exceed an order of magnitude. The effect can be employed in fabrication of inexpensive DNA sensors.
| S-EPMC2729444 | biostudies-literature
Project description:Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K+, Na+, Cs+ and Li+; organic cations: TMA and TEA, and divalents: Ba2+, Ca2+, Mg2+ and Mn2+. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs+, Na+ and K+ had chord conductances in the range of 35-55 pS with the exception of Li+, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K+, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba2+ and 15 pS for Ca2+ at -80 mV and 10 pS for Mg2+ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K+ currents much like the divalents. As expected, the channel K+ conductance increased with K+ concentration saturating at ~ 45 pS and the KD of K+ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.
| S-EPMC4425559 | biostudies-literature
Project description:The neuronal subthreshold-operating A-type K(+) current regulates electrical excitability, spike timing, and synaptic integration and plasticity. The Kv4 channels underlying this current have been implicated in epilepsy, regulation of dopamine release, and pain plasticity. However, the unitary conductance (gamma) of neuronal somatodendritic A-type K(+) channels composed of Kv4 pore-forming subunits is larger (approximately 7.5 pS) than that of Kv4 channels expressed singly in heterologous cells (approximately 4 pS). Here, we examined the putative novel contribution of the dipeptidyl-peptidase-like protein-6 DPP6-S to the gamma of native [cerebellar granule neuron (CGN)] and reconstituted Kv4.2 channels. Coexpression of Kv4.2 proteins with DPP6-S was sufficient to match the gamma of native CGN channels; and CGN Kv4 channels from dpp6 knock-out mice yielded a gamma indistinguishable from that of Kv4.2 channels expressed singly. Moreover, suggesting electrostatic interactions, charge neutralization mutations of two N-terminal acidic residues in DPP6-S eliminated the increase in gamma. Therefore, DPP6-S, as a membrane protein extrinsic to the pore domain, is necessary and sufficient to explain a fundamental difference between native and recombinant Kv4 channels. These observations may help to understand the molecular basis of neurological disorders correlated with recently identified human mutations in the dpp6 gene.
| S-EPMC3758885 | biostudies-other
Project description:Gap junction (GJ) channels provide direct passage for ions and small molecules to be exchanged between neighbouring cells and are crucial for many physiological processes. GJ channels can be gated by transjunctional voltage (known as Vj-gating) and display a wide range of unitary channel conductance (γj), yet the domains responsible for Vj-gating and γj are not fully clear. The first extracellular domain (E1) of several connexins has been shown to line part of their GJ channel pore and play important roles in Vj-gating properties and/or ion permeation selectivity. To test roles of the E1 of Cx50 GJ channels, we generated a chimera, Cx50Cx36E1, where the E1 domain of Cx50 was replaced with that of Cx36, a connexin showing quite distinct Vj-gating and γj from those of Cx50. Detailed characterizations of the chimera and three point mutants in E1 revealed that, although the E1 domain is important in determining γj, the E1 domain of Cx36 is able to effectively function within the context of the Cx50 channel with minor changes in Vj-gating properties, indicating that sequence differences between the E1 domains in Cx36 and Cx50 cannot account for their drastic differences in Vj-gating and γj. Our homology models of the chimera and the E1 mutants revealed that electrostatic properties of the pore-lining residues and their contribution to the electric field in the pore are important factors for the rate of ion permeation of Cx50 and possibly other GJ channels.
| S-EPMC4666595 | biostudies-literature
Project description:Destructive quantum interference has been shown to strongly reduce charge tunneling rates across molecular bridges. The current consensus is that destructive quantum interference occurs in cross-conjugated molecules, while linearly conjugated molecules exhibit constructive interference. Our experimental results on photoinduced charge transfer in donor-bridge-acceptor systems, however, show that hole transfer is ten times faster through a cross-conjugated biphenyl bridge than through a linearly conjugated biphenyl bridge. Electronic structure calculations reveal that the surprisingly low hole transfer rate across the linearly conjugated biphenyl bridge is caused by the presence of destructive instead of constructive interference. We find that the specific molecular orbital symmetry of the involved donor and acceptor states leads to interference conditions that are different from those valid in single molecule conduction experiments. Furthermore, the results indicate that by utilizing molecular orbital symmetry in a smart way new opportunities of engineering charge transfer emerge.
| S-EPMC5707500 | biostudies-other
Project description:The electrochemical gradients established across cell membranes are paramount for the execution of biological functions. Besides ion channels, other transporters, such as exogenous pore-forming toxins, may present ionic selectivity upon reconstitution in natural and artificial lipid membranes and contribute to the electrochemical gradients. In this context, we utilized electrophysiology approaches to assess the ionic selectivity of the pore-forming toxin lysenin reconstituted in planar bilayer lipid membranes. The membrane voltages were determined from the reversal potentials recorded upon channel exposure to asymmetrical ionic conditions, and the permeability ratios were calculated from the fit with the Goldman-Hodgkin-Katz equation. Our work shows that lysenin channels are ion-selective and the determined permeability coefficients are cation and anion-species dependent. We also exploited the unique property of lysenin channels to transition to a stable sub-conducting state upon exposure to calcium ions and assessed their subsequent change in ionic selectivity. The observed loss of selectivity was implemented in an electrical model describing the dependency of reversal potentials on calcium concentration. In conclusion, our work demonstrates that this pore-forming toxin presents ionic selectivity but this is adjusted by the particular conduction state of the channels.
| S-EPMC8622276 | biostudies-literature
Project description:Voltage-gated sodium channels (NaVs) play fundamental roles in eukaryotes, but their exceptional size hinders their structural resolution. Bacterial NaVs are simplified homologues of their eukaryotic counterparts, but their use as models of eukaryotic Na+ channels is limited by their homotetrameric structure at odds with the asymmetric Selectivity Filter (SF) of eukaryotic NaVs. This work aims at mimicking the SF of eukaryotic NaVs by engineering radial asymmetry into the SF of bacterial channels. This goal was pursued with two approaches: the co-expression of different monomers of the NaChBac bacterial channel to induce the random assembly of heterotetramers, and the concatenation of four bacterial monomers to form a concatemer that can be targeted by site-specific mutagenesis. Patch-clamp measurements and Molecular Dynamics simulations showed that an additional gating charge in the SF leads to a significant increase in Na+ and a modest increase in the Ca2+ conductance in the NavMs concatemer in agreement with the behavior of the population of random heterotetramers with the highest proportion of channels with charge -5e. We thus showed that charge, despite being important, is not the only determinant of conduction and selectivity, and we created new tools extending the use of bacterial channels as models of eukaryotic counterparts.
| S-EPMC7764494 | biostudies-literature
Project description:The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO42-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 A from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 A, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.
| S-EPMC2229543 | biostudies-literature
Project description:Electrophysiological characterization of large protein channels, usually displaying multi-ionic transport and weak ion selectivity, is commonly performed at physiological conditions (moderate gradients of KCl solutions at decimolar concentrations buffered at neutral pH). We extend here the characterization of the OmpF porin, a wide channel of the outer membrane of E. coli, by studying the effect of salts of divalent cations on the transport properties of the channel. The regulation of divalent cations concentration is essential in cell metabolism and understanding their effects is of key importance, not only in the channels specifically designed to control their passage but also in other multiionic channels. In particular, in porin channels like OmpF, divalent cations modulate the efficiency of molecules having antimicrobial activity. Taking advantage of the fact that the OmpF channel atomic structure has been resolved both in water and in MgCl(2) aqueous solutions, we analyze the single channel conductance and the channel selectivity inversion aiming to separate the role of the electrolyte itself, and the counterion accumulation induced by the protein channel charges and other factors (binding, steric effects, etc.) that being of minor importance in salts of monovalent cations become crucial in the case of divalent cations.
| S-EPMC3449104 | biostudies-literature