scholarly journals Localization of the gate and selectivity filter of the full-length P2X7 receptor

2017 ◽  
Vol 114 (11) ◽  
pp. E2156-E2165 ◽  
Author(s):  
Anja Pippel ◽  
Michaela Stolz ◽  
Ronja Woltersdorf ◽  
Achim Kless ◽  
Günther Schmalzing ◽  
...  
Keyword(s):  
P2x7 Receptor ◽  
Single Channel ◽  
Ion Selectivity ◽  
Selectivity Filter ◽  
Transmembrane Helices ◽  
Transmembrane Channel ◽  
Channel Opening ◽  
Inflammatory Processes ◽  
P2x4 Receptors ◽  
Single Channel Currents

The P2X7 receptor (P2X7R) belongs to the P2X family of ATP-gated cation channels. P2X7Rs are expressed in epithelial cells, leukocytes, and microglia, and they play important roles in immunological and inflammatory processes. P2X7Rs are obligate homotrimers, with each subunit having two transmembrane helices, TM1 and TM2. Structural and functional data regarding the P2X2 and P2X4 receptors indicate that the central trihelical TM2 bundle forms the intrinsic transmembrane channel of P2X receptors. Here, we studied the accessibility of single cysteines substituted along the pre-TM2 and TM2 helix (residues 327–357) of the P2X7R using as readouts (i) the covalent maleimide fluorescence accessibility of the surface-bound P2X7R and (ii) covalent modulation of macroscopic and single-channel currents using extracellularly and intracellularly applied methanethiosulfonate (MTS) reagents. We found that the channel opening extends from the pre-TM2 region through the outer half of the trihelical TM2 channel. Covalently adducted MTS ethylammonium+ (MTSEA+) strongly increased the probability that the channel was open by delaying channel closing of seven of eight responsive human P2X7R (hP2X7R) mutants. Structural modeling, as supported by experimental probing, suggested that resulting intraluminal hydrogen bonding interactions stabilize the open-channel state. The additional decrease in single-channel conductance by MTSEA+ in five of seven positions identified Y336, S339, L341C, Y343, and G345 as the narrowest part of the channel lumen. The gate and ion-selectivity filter of the P2X7R could be colocalized at and around residue S342. None of our results provided any evidence for dilation of the hP2X7R channel on sustained stimulation with ATP4−.

scholarly journals Conformational Changes in the Pore of CLC-0

2003 ◽  
Vol 122 (3) ◽  
pp. 277-294 ◽  
Author(s):  
Alessio Accardi ◽  
Michael Pusch
Keyword(s):  
Conformational Changes ◽  
Single Channel ◽  
General Structure ◽  
Point Mutations ◽  
Clofibric Acid ◽  
Selectivity Filter ◽  
Side Chain ◽  
Closed State ◽  
State Dependent ◽  
Channel Opening

The Torpedo Cl− channel, CLC-0, is inhibited by clofibric acid derivatives from the intracellular side. We used the slow gate-deficient mutant CLC-0C212S to investigate the mechanism of block by the clofibric acid–derivative p-chlorophenoxy-acetic acid (CPA). CPA blocks open channels with low affinity (KDO= 45 mM at 0 mV) and shows fast dissociation (koff = 490 s−1 at −140 mV). In contrast, the blocker binds to closed channels with higher affinity and with much slower kinetics. This state-dependent block coupled with the voltage dependence of the gating transitions results in a highly voltage-dependent inhibition of macroscopic currents (KD ∼1 mM at −140 mV; KD ∼65 mM at 60 mV). The large difference in CPA affinity of the open and closed state suggests that channel opening involves more than just a local conformational rearrangement. On the other hand, in a recent work (Dutzler, R., E.B. Campbell, and R. MacKinnon. 2003. Science. 300:108–112) it was proposed that the conformational change underlying channel opening is limited to a movement of a single side chain. A prediction of this latter model is that mutations that influence CPA binding to the channel should affect the affinities for an open and closed channel in a similar manner since the general structure of the pore remains largely unchanged. To test this hypothesis we introduced point mutations in four residues (S123, T471, Y512, and K519) that lie close to the intracellular pore mouth or to the putative selectivity filter. Mutation T471S alters CPA binding exclusively to closed channels. Pronounced effects on the open channel block are observed in three other mutants, S123T, Y512A, and K519Q. Together, these results collectively suggest that the structure of the CPA binding site is different in the open and closed state. Finally, replacement of Tyr 512, a residue directly coordinating the central Cl− ion in the crystal structure, with Phe or Ala has very little effect on single channel conductance and selectivity. These observations suggest that channel opening in CLC-0 consists in more than a movement of a side chain and that other parts of the channel and of the selectivity filter are probably involved.

scholarly journals Divalent cation activation and inhibition of single calcium release channels from sheep cardiac sarcoplasmic reticulum.

1990 ◽  
Vol 95 (5) ◽  
pp. 981-1005 ◽  
Author(s):  
R H Ashley ◽  
A J Williams
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Calcium Release ◽  
Single Channel ◽  
Fluctuation Analysis ◽  
Open Probability ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Channel Opening ◽  
Lifetime Analysis ◽  
Single Channel Currents

Single Ca2+ release channels from vesicles of sheep cardiac junctional sarcoplasmic reticulum have been incorporated into uncharged planar lipid bilayers. Single-channel currents were recorded from Ca2(+)-activated channels that had a Ca2+ conductance of approximately 90 pS. Channel open probability increased sublinearly as the concentration of free Ca2+ was raised at the myoplasmic face, and without additional agonists the channels could not be fully activated even by 100 microM free Ca2+. Lifetime analysis revealed a minimum of two open and three closed states, and indicates that Ca2+ activated the channels by interacting with at least one of the closed states to increase the rate of channel opening. Correlations between adjacent lifetimes suggested there were at least two pathways between the open- and closed-state aggregates. An analysis of bursting behavior also revealed correlations between successive burst lengths and the number of openings per burst. The latter had two geometric components, providing additional evidence for at least two open states. One component appeared to comprise unit bursts, and the lifetime of most of these fell within the dominant shorter open-time distribution associated with over 90% of all openings. A cyclic gating scheme is proposed, with channel activation regulated by the binding of Ca2+ to a closed conformation of the channel protein. Mg2+ may inhibit activation by competing for this binding site, but lifetime and fluctuation analysis suggested that once activated the channels continue to gate normally.

scholarly journals Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices

2022 ◽  
Author(s):  
Nurunisa Akyuz ◽  
K. Domenica Karavitaki ◽  
Bifeng Pan ◽  
Panos I. Tamvakologos ◽  
Kelly P. Brock ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Hair Cells ◽  
Single Channel ◽  
Transmembrane Helices ◽  
Open Probability ◽  
Transmembrane Channel ◽  
Force Sensitivity ◽  
Electrophysiological Recordings ◽  
Auditory Transduction ◽  
Pore Lining

The transmembrane channel-like (TMC) 1 and 2 proteins play a central role in auditory transduction, forming ion channels that convert sound into electrical signals. However, the molecular mechanism of their gating remains unknown. Here, using predicted structural models as a guide, we probed the effects of twelve mutations on the mechanical gating of the transduction currents in native hair cells of Tmc1/2-null mice expressing virally introduced TMC1 variants. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining transmembrane (TM) helices 4 and 6 modified gating, reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel.

Na+-induced inward rectification in the two-pore domain K+ channel, TASK-2

2005 ◽  
Vol 288 (1) ◽  
pp. F162-F169 ◽  
Author(s):  
Michael J. Morton ◽  
Sarah Chipperfield ◽  
Abdulrahman Abohamed ◽  
Asipu Sivaprasadarao ◽  
Malcolm Hunter
Keyword(s):  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Selectivity Filter ◽  
Inward Rectification ◽  
K Channel ◽  
Open Probability ◽  
Whole Cell ◽  
Single Channel Currents ◽  
Pore Domain

TASK-2 is a member of the two-pore domain K+ (K2P) channel family that is expressed at high levels in several epithelia, including the proximal tubule. In common with the other TASK channels, TASK-2 is sensitive to changes in extracellular pH. We have expressed human TASK-2 in Chinese hamster ovary cells and studied whole cell and single-channel activity by patch clamp. The open probability of K2P channels is generally independent of voltage, yielding linear current-voltage ( I- V) curves. Despite these properties, we found that these channels showed distinct inward rectification immediately on the establishment of whole cell clamp, which became progressively less pronounced with time. This rectification was due to intracellular Na+ but was unaffected by polyamines or Mg2+ (agents that cause rectification in Kir channels). Rectification was concentration- and voltage-dependent and could be reversibly induced by switching between Na+-rich and Na+-free bath solutions. In excised inside-out patches, Na+ reduced the amplitude of single-channel currents, indicative of rapid block and unblock of the pore. Mutations in the selectivity filter abolished Na+-induced rectification, suggesting that Na+ binds within the selectivity filter in wild-type channels. This sensitivity to intracellular Na+ may be an additional potential regulatory mechanism of TASK-2 channels.

scholarly journals Tail End of the S6 Segment

2002 ◽  
Vol 120 (1) ◽  
pp. 87-97 ◽  
Author(s):  
Shinghua Ding ◽  
Richard Horn
Keyword(s):  
Potassium Channels ◽  
Energy Barrier ◽  
Single Channel ◽  
Noise Analysis ◽  
Selectivity Filter ◽  
Open Probability ◽  
Cytoplasmic Extension ◽  
Activation Gate ◽  
Single Channel Currents ◽  
Channel Currents

The permeation pathway in voltage-gated potassium channels has narrow constrictions at both the extracellular and intracellular ends. These constrictions might limit the flux of cations from one side of the membrane to the other. The extracellular constriction is the selectivity filter, whereas the intracellular bundle crossing is proposed to act as the activation gate that opens in response to a depolarization. This four-helix bundle crossing is composed of S6 transmembrane segments, one contributed by each subunit. Here, we explore the cytoplasmic extension of the S6 transmembrane segment of Shaker potassium channels, just downstream from the bundle crossing. We substituted cysteine for each residue from N482 to T489 and determined the amplitudes of single channel currents and maximum open probability (Po,max) at depolarized voltages using nonstationary noise analysis. One mutant, F484C, significantly reduces Po,max, whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (γ). Mutations of residue Y485 have no effect on the Rb+/K+ selectivity, suggesting a local effect on γ rather than an allosteric effect on the selectivity filter. Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate. Replacing Rb+ ions for K+ ions reduces the amplitude of single channel currents and makes γ insensitive to mutations of Y485. These results suggest that Rb+ ions increase an extracellular energy barrier, presumably at the selectivity filter, thus making it rate limiting for flux of permeant ions. These results indicate that S6T residues have an influence on the conformation of the open activation gate, reflected in both the stability of the open state and the energy barriers it presents to ions.

scholarly journals Aromatic–aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process

2014 ◽  
Vol 143 (2) ◽  
pp. 289-307 ◽  
Author(s):  
Line Garneau ◽  
Hélène Klein ◽  
Marie-France Lavoie ◽  
Emmanuelle Brochiero ◽  
Lucie Parent ◽  
...  
Keyword(s):  
Selectivity Filter ◽  
Transmembrane Helices ◽  
Open Probability ◽  
Channel Pore ◽  
Transmembrane Segments ◽  
Aromatic Interactions ◽  
C Terminus ◽  
Helical Segment ◽  
Channel Opening ◽  
Dynamics Simulations

The Ca2+-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca2+ concentrations (Pomax) is low, typically 0.1–0.2 for KCa3.1 wild type. This observation argues for the binding of Ca2+ to the calmodulin (CaM)–KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca2+-dependent gating of KCa3.1 originates from the binding of Ca2+ to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic–aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic–aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators.

scholarly journals Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels

2004 ◽  
Vol 124 (6) ◽  
pp. 729-739 ◽  
Author(s):  
Yu-Yang Zhang ◽  
Janice L. Robertson ◽  
Daniel A. Gray ◽  
Lawrence G. Palmer
Keyword(s):  
Single Channel ◽  
Ion Selectivity ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
Single Amino Acid ◽  
Structural Basis ◽  
K Channels ◽  
Chord Conductance ◽  
In Series ◽  
Carboxy Terminal

Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at Vm = −100 mV from 34 to 21 pS. The slope conductance measured between −60 and −140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba2+, suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl+:K+, accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K+ transport.

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