Controlling Ion Permeation Research Articles

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.1

Irritant-evoked activation and calcium modulation of the TRPA1 receptor.

The TRPA1 ion channel is expressed by primary afferent nerve fibers, where it functions as a low threshold sensor for structurally diverse electrophilic irritants ranging from small volatile environmental toxicants to endogenous algogenic lipids1. TRPA1 is also a ‘receptor-operated’ channel whose activation downstream of metabotropic receptors elicits inflammatory pain or itch, making it an attractive target for novel analgesic therapies2. However, we lack mechanistic insight into how TRPA1 recognizes and responds to electrophiles or cytoplasmic second messengers. Here, we show that electrophiles act through a two-step process in which modification of a highly reactive cysteine (C621) promotes reorientation of a cytoplasmic loop to enhance nucleophilicity and modification of a nearby cysteine (C665), thereby stabilizing the loop in an activating configuration. These actions modulate two restrictions controlling ion permeation, including widening of the selectivity filter to enhance calcium permeability and opening of a canonical gate at the cytoplasmic end of the pore. We propose a model to explain functional coupling between electrophile action and these control points. We also characterize a calcium binding pocket that is remarkably conserved across TRP channel subtypes and accounts for all aspects of calcium-dependent TRPA1 regulation, including potentiation, desensitization, and activation by metabotropic receptors. These findings provide a structural framework for understanding how a broad-spectrum irritant receptor is controlled by endogenous and exogenous agents that elicit or exacerbate pain and itch.

The Outer Pore and Selectivity Filter of TRPA1.

TRPA1 (transient-receptor-potential-related ion channel with ankyrin domains) is a direct receptor or indirect effector for a wide variety of nociceptive signals, and thus is a compelling target for development of analgesic pharmaceuticals such as channel blockers. Recently, the structure of TRPA1 was reported, providing insights into channel assembly and pore architecture. Here we report whole-cell and single-channel current recordings of wild-type human TRPA1 as well as TRPA1 bearing point mutations of key charged residues in the outer pore. These measurements demonstrate that the glutamate at position 920 plays an important role in collecting cations into the mouth of the pore, by changing the effective surface potential by ~16 mV, while acidic residues further out have little effect on permeation. Electrophysiology experiments also confirm that the aspartate residue at position 915 represents a constriction site of the TRPA1 pore and is critical in controlling ion permeation.

The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel.

Inwardly rectifying K+ (Kir) channels, serving as natural molecular nanomachines, transport potassium ions across the plasma membrane of the cell. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter (SF), the inner helix bundle crossing (HBC), and the cytosolic G loop) may serve as gates to control ion permeation. Our previous molecular dynamics simulations based on the crystal structure of a Kir3.1 chimera revealed the possible gating mechanism of the G loop gate. Here, we introduced a proline mutation in the inner helix and obtained a channel model of the open HBC gate. The open HBC gate reaches 0.6 nm in diameter, which allows partial hydrated K+ ions to pass through. During the gating process, both the transmembrane helices TM1 and TM2 cooperatively rotate in a counterclockwise direction (viewed from the extracellular side) with the aid of the phospholipid PIP2. Only when all the transmembrane helices adopt a counterclockwise rotation, the HBC gate can be stabilized in the open state. We estimate that introduction of the proline mutation decreases the energy required to open the HBC gate by about 1.4 kcal/mol (ΔΔG).

Simulations of the Helix Bundle Crossing Gate Opening in Kir Channels

Inwardly rectifying K+ (Kir) channels are gated by the phospholipid PIP2. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter -SF, the inner helix bundle crossing (HBC), and the intracellular G-loop) may serve as gates to control ion permeation. A crystal structure of a Kir3.1 chimera [Nishida et al., 2007] captured the cytosolic G-loop gate in “closed/constricted” or “open/dilated” conformations. 100 ns Molecular Dynamics (MD) simulations studying the PIP2-driven Kir channel activation of the Kir3.1 chimera led us to propose a molecular mechanism of the G-loop gate opening [Meng et al., 2012]. However, opening of the HBC gate was not observed throughout this simulation. Mutagenesis and single-channel recording studies in our lab showed that a proline mutation on the inner helix of the Kir3.4 channel dramatically increased the open probability of the channel [Jin et al., 2002]. We introduced the corresponding M170P mutation on the Kir3.1 chimera structure and ran 100 ns long simulations of four mutant channel systems: dilated and constricted M170P Kir3.1 chimera in the presence (holo) and absence (apo) of PIP2, using the GROMACS program [Hess et al., 2008]. Three potassium ions present in the SF passed through the HBC gate in the system of the holo dilated M170P Kir3.1 chimera within the 100 ns simulation time. Minimal distance measurements indicated that the HBC gate was able to open only when PIP2 was present and the G-loop gate was stabilized in the open state. Principal component analysis revealed coupled conformational changes in the Slide helix, DE- and LM-loops, possibly related to the opening of the HBC gate. Moreover, unique residue interactions within the transmembrane domains were observed in the dilated holo system. Predictions of these models are being tested experimentally.

Reconstruction of the P2X2 Receptor Reveals a Vase-Shaped Structure with Lateral Tunnels above the Membrane

In response to the intercellular messenger ATP, P2X receptors transfer various sensory information, including pain. Here we have reconstructed the structure of the P2X(2) receptor at 15 A resolution from more than 90,000 particle images, taken with a cryo-electron microscope equipped with a helium-cooled stage. This three-dimensional depiction, presumably in a closed state, revealed an elongated vase-shaped structure 202 A in height and 160 A in major diameter. The extracellular and transmembrane domains present a two-layered structure, in which a sparse outer layer surrounds a pore-forming inner density. The decreased diameter of a putative ion-conducting pathway at the middle of the membrane was considered to be the narrowest part of the pore, which has been predicted from electrophysiological studies. The sparse, extended structure of the P2X(2) receptor indicates a loose assembly of subunits, which could be a basis for the activation-dependent pore dilation of P2X receptors.

Potential of Mean Force Calculations of Ion Permeation in Gramicidin A Channel

The potential of mean force (PMF) for K+ ion permeation through the gramicidin A (gA) channel were calculated from the molecular dynamics (MD) simulations with four different force fields (FF): CHARMM27, CHARMM27 with dihedral-based correction map (CHARMM27+CMAP), CHARMM27 with a improved FF parameters for tryptophan indole ring (CHARMM27+Trp), and CHARMM27 with the CMAP and the improved FF parameters for Trp (CHARMM27+CMAP+Trp). When comparing the PMFs obtained with these four different FF, we find that both CHARMM27 and CHARMM27+Trp predict free energy profiles that are in semi-quantitative agreement with measurements of the conductance and dissociation constant. The combination CHARMM27+Trp gives the best agreement. However, the CHARMM27+CMAP yields a larger barrier in the PMF and CHARMM27+CMAP+Trp generates a deeper binding potential well. These calculations illustrate the sensitivity of the PMF controlling ion permeation to subtle changes in the FF. We also compute a 2-ion PMF for a doubly occupied gA channel. The effect of the number of water molecules in the channel on the effective ion-ion interactions is also studied. Elucidating the properties of the doubly occupied channel is important because experiments are often carried out at fairly high concentration where double ion occupancy is predominant.

Poly(N‐isopropylacrylamide) Interfaces with Dissimilar Thermo‐responsive Behavior for Controlling Ion Permeation and Immobilization

AbstractStimuli‐responsive polymer interfaces with tethered poly(N‐isopropylacrylamide) (PNIPAm) chains and its cross‐linked porous hydrogel as sensitive phases were fabricated by cycling an electronic potential in aqueous solution. Surface morphological analysis and electrochemical measurements revealed that, owing to their different novel structures, the PNIPAm chain‐modified interface showed ON/OFF switch behavior, whereas the PNIPAm gel‐modified interface exhibited a ‘breathing in' process. The results suggest that the interfacial physicochemical properties, which are greatly affected by the graft conformations and topologies of PNIPAm on the substrates, could be effectively modulated by easily varying the synthesis conditions and are investigated by simple electrochemical methods. The results also demonstrate that the ON/OFF switch behavior of the PNIPAm chain‐modified interface has potential applications in controlled ion/molecule permeation, and the ‘breathing in' mechanism of the gel‐modified interface might be applied to immobilize ions or nanoparticles. These thermo‐sensitive interfaces might be used to design adaptive/responsive biocompatible surfaces in a variety of areas.

A Molecular Determinant of Nickel Inhibition in Cav3.2 T-type Calcium Channels

Molecular cloning studies have revealed that heterogeneity of T-type Ca2+ currents in native tissues arises from the three isoforms of Ca(v)3 channels: Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3. From pharmacological analysis of the recombinant T-type channels, low concentrations (<50 microM) of nickel were found to selectively block the Ca(v)3.2 over the other isoforms. To date, however, the structural element(s) responsible for the nickel block on the Ca(v)3.2 T-type Ca2+ channel remain unknown. Thus, we constructed chimeric channels between the nickel-sensitive Ca(v)3.2 and the nickel-insensitive Ca(v)3.1 to localize the region interacting with nickel. Systematic assaying of serial chimeras suggests that the region preceding domain I S4 of Ca(v)3.2 contributes to nickel block. Point mutations of potential nickel-interacting sites revealed that H191Q in the S3-S4 loop of domain I significantly attenuated the nickel block of Ca(v)3.2, mimicking the nickel-insensitive blocking potency of Ca(v)3.1. These findings indicate that His-191 in the S3-S4 loop is a critical residue conferring nickel block to Ca(v)3.2 and reveal a novel role for the S3-S4 loop to control ion permeation through T-type Ca2+ channels.

Heteromeric AMPA Receptors Assemble with a Preferred Subunit Stoichiometry and Spatial Arrangement

AMPA receptors are thought to be a tetrameric assembly of the subunits GluR1-4. We have examined whether two coexpressed subunits (GluR1/2) combine at random to form channels, or preferentially assemble with a specific stoichiometry and spatial configuration. The subunits carried markers controlling ion permeation and desensitization, and these properties were monitored as a function of relative expression level and subunit composition. Homomeric receptors assembled stochastically while heteromeric receptors preferentially formed with a stoichiometry of two GluR1 and two GluR2 subunits, and with identical subunits positioned on opposite sides of the channel pore. This structure will predominate if GluR1 binds to GluR2 more rapidly during receptor assembly than other subunit combinations. The practical outcome of selective heteromeric assembly is a more homogenous receptor population in vivo.

Bilayer lipid membranes for electrochemical sensing

AbstractBilayer lipid membranes (BLMs) are described as selective and sensitive electrochemical transducers for inorganic ions and organic species. The electrostatic properties of BLMs, which are significant in controlling ion permeation processes, and the possible mechanisms for signal generation are presented herein. Operation of a BLM‐based transducer hinges on a selective interaction between analyte in aqueous solution and membrane‐embedded receptors; as a result, the electrostatic fields and/or the physical phase structure of the membrane alters, leading to an analytical signal (most commonly conductivity changes) which can be related to the analyte concentration. Examples of electrochemical sensors based on this principle and efforts to stabilize these bilayer membranes are presented in this review article.

Role of H5 domain in determining pore diameter and ion permeation through cyclic nucleotide-gated channels

Ion permeation through membrane channels is thought to be governed by a narrow region of the channel pore termed the selectivity filter, which has been proposed to discriminate among ions by both specific binding and molecular sieving, as determined by pore diameter. Recent evidence suggests that a conserved domain (known as H5, P or SS1-SS2) in voltage-gated potassium, sodium and calcium channels contributes to the lining of the pore. Here we investigate whether the H5 domain determines pore diameter and examine the role of pore diameter in controlling ion permeation. These studies rely on differences in single channel conductance, ion selectivity and apparent pore diameter between cyclic nucleotide-gated channels cloned from bovine retina and catfish olfactory neurons. Using chimaeric retinal-olfactory channels, we find that the H5 domain determines these differences in permeation properties, providing structural evidence that the cyclic nucleotide-gated channels are indeed members of the voltage-gated channel family. Moreover, these results show directly that the H5 domain helps form the selectivity filter and that molecular sieving is important in controlling ion permeation.

Identification of a site in glutamate receptor subunits that controls calcium permeability.

The neurotransmitter glutamate mediates excitatory synaptic transmission throughout the brain. A family of genes encoding subunits of the non-N-methyl-D-aspartate (non-NMDA) type of glutamate receptor has been cloned. Some combinations of these subunits assemble into receptors with a substantial permeability to calcium, whereas others do not. To investigate the structural features that control ion permeation through these ligand-gated channels, mutant receptor subunits with single-amino acid changes were constructed. Mutation of a certain amino acid that results in a net charge change (from glutamine to arginine or vice versa) alters both the current-voltage relation and the calcium permeability of non-NMDA receptors. A site has thus been identified that regulates the permeation properties of these glutamate receptors.