scholarly journals Energetic and Spatial Parameters for Gating of the Bacterial Large Conductance Mechanosensitive Channel, MscL

1999 ◽  
Vol 113 (4) ◽  
pp. 525-540 ◽  
Author(s):  
Sergei I. Sukharev ◽  
Wade J. Sigurdson ◽  
Ching Kung ◽  
Frederick Sachs
Keyword(s):  
Conformational Changes ◽  
Single Channel ◽  
Energy Difference ◽  
Boltzmann Distribution ◽  
Mechanosensitive Channel ◽  
Radius Of Curvature ◽  
Closed State ◽  
Spatial Parameters ◽  
Single Channel Currents ◽  
Plane Area

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was ΔE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (ΔA), the free energy, TΔA, would correspond to ΔA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest ΔA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to ΔA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.

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 Single Channel Properties of P2X2 Purinoceptors

1999 ◽  
Vol 113 (5) ◽  
pp. 695-720 ◽  
Author(s):  
Shinghua Ding ◽  
Frederick Sachs
Keyword(s):  
Conformational Changes ◽  
Single Channel ◽  
Excess Noise ◽  
Hill Coefficient ◽  
Inward Rectification ◽  
Channel Current ◽  
Single Channel Current ◽  
The Mean ◽  
Single Channel Currents ◽  
Channel Properties

The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current–voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of ∼30 pS at −100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was ∼150 mM at 0 mV and voltage dependent. The binding site appeared to be ∼0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 μM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of ∼7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose–response curve.

scholarly journals NMDA receptor channel gating control by the pre-M1 helix

2020 ◽  
Vol 152 (4) ◽  
Author(s):  
Miranda J. McDaniel ◽  
Kevin K. Ogden ◽  
Steven A. Kell ◽  
Pieter B. Burger ◽  
Dennis C. Liotta ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Conformational Changes ◽  
Time Course ◽  
Transmembrane Domain ◽  
Single Channel ◽  
De Novo ◽  
Channel Gating ◽  
Open Probability ◽  
Single Channel Currents

The NMDA receptor (NMDAR) is an ionotropic glutamate receptor formed from the tetrameric assembly of GluN1 and GluN2 subunits. Within the flexible linker between the agonist binding domain (ABD) and the M1 helix of the pore-forming transmembrane helical bundle lies a two-turn, extracellular pre-M1 helix positioned parallel to the plasma membrane and in van der Waals contact with the M3 helix thought to constitute the channel gate. The pre-M1 helix is tethered to the bilobed ABD, where agonist-induced conformational changes initiate activation. Additionally, it is a locus for de novo mutations associated with neurological disorders, is near other disease-associated de novo sites within the transmembrane domain, and is a structural determinant of subunit-selective modulators. To investigate the role of the pre-M1 helix in channel gating, we performed scanning mutagenesis across the GluN2A pre-M1 helix and recorded whole-cell macroscopic and single channel currents from HEK293 cell-attached patches. We identified two residues at which mutations perturb channel open probability, the mean open time, and the glutamate deactivation time course. We identified a subunit-specific network of aromatic amino acids located in and around the GluN2A pre-M1 helix to be important for gating. Based on these results, we are able to hypothesize about the role of the pre-M1 helix in other NMDAR subunits based on sequence and structure homology. Our results emphasize the role of the pre-M1 helix in channel gating, implicate the surrounding amino acid environment in this mechanism, and suggest unique subunit-specific contributions of pre-M1 helices to GluN1 and GluN2 gating.

scholarly journals A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII

2021 ◽  
Vol 118 (32) ◽  
pp. e2108967118
Author(s):  
Ximena López ◽  
Nicolás Palacios-Prado ◽  
Juan Güiza ◽  
Rosalba Escamilla ◽  
Paola Fernández ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Uptake Rate ◽  
Single Channel ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Membrane Stretch ◽  
C Terminus ◽  
Putative Phosphorylation Site ◽  
In Silico Studies ◽  
Single Channel Currents

Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation—measured by changes in DAPI uptake rate—was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.

scholarly journals Shaker potassium channel gating. III: Evaluation of kinetic models for activation.

1994 ◽  
Vol 103 (2) ◽  
pp. 321-362 ◽  
Author(s):  
W N Zagotta ◽  
T Hoshi ◽  
R W Aldrich
Keyword(s):  
Steady State ◽  
Conformational Changes ◽  
Time Course ◽  
Single Channel ◽  
Ionic Currents ◽  
Single Step ◽  
Gating Currents ◽  
Open Probability ◽  
Shaker Potassium Channel ◽  
Single Channel Currents

Predictions of different classes of gating models involving identical conformational changes in each of four subunits were compared to the gating behavior of Shaker potassium channels without N-type inactivation. Each model was tested to see if it could simulate the voltage dependence of the steady state open probability, and the kinetics of the single-channel currents, macroscopic ionic currents and macroscopic gating currents using a single set of parameters. Activation schemes based upon four identical single-step activation processes were found to be incompatible with the experimental results, as were those involving a concerted, opening transition. A model where the opening of the channel requires two conformational changes in each of the four subunits can adequately account for the steady state and kinetic behavior of the channel. In this model, the gating in each subunit is independent except for a stabilization of the open state when all four subunits are activated, and an unstable closed conformation that the channel enters after opening. A small amount of negative cooperativity between the subunits must be added to account quantitatively for the dependence of the activation time course on holding voltage.

scholarly journals Correlation of membrane protein conformational and functional dynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Raghavendar Reddy Sanganna Gari ◽  
Joel José Montalvo‐Acosta ◽  
George R. Heath ◽  
Yining Jiang ◽  
Xiaolong Gao ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Conformational Changes ◽  
Single Channel ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
High Temporal Resolution ◽  
Dependent Manner ◽  
Functional Dynamics ◽  
Ph Dependent ◽  
Open States

AbstractConformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

scholarly journals Effects of Strontium on the Permeation and Gating Phenotype of Calcium Channels in Hair Cells

2008 ◽  
Vol 100 (4) ◽  
pp. 2115-2124 ◽  
Author(s):  
Adrian Rodriguez-Contreras ◽  
Ping Lv ◽  
Jun Zhu ◽  
Hyo Jeong Kim ◽  
Ebenezer N. Yamoah
Keyword(s):  
Hair Cell ◽  
Single Channel ◽  
Single Channels ◽  
Physiological Concentration ◽  
Closed State ◽  
Dependent Inactivation ◽  
Intracellular Ca ◽  
High Concentrations ◽  
Latency Distributions ◽  
Channel Properties

To minimize the effects of Ca2+ buffering and signaling, this study sought to examine single Ca2+ channel properties using Sr2+ ions, which substitute well for Ca2+ but bind weakly to intracellular Ca2+ buffers. Two single-channel fluctuations were distinguished by their sensitivity to dihydropyridine agonist (L-type) and insensitivity toward dihydropyridine antagonist (non-L-type). The L- and non-L-type single channels were observed with single-channel conductances of 16 and 19 pS at 70 mM Sr2+ and 11 and 13 pS at 5 mM Sr2+, respectively. We obtained KD estimates of 5.2 and 1.9 mM for Sr2+ for L- and non-L-type channels, respectively. At Ca2+ concentration of ∼2 mM, the single-channel conductances of Sr2+ for the L-type channel was ∼1.5 and 4.0 pS for the non-L-type channels. Thus the limits of single-channel microdomain at the membrane potential of a hair cell (e.g., −65 mV) for Sr2+ ranges from 800 to 2,000 ion/ms, assuming an ECa of 100 mV. The channels are ≥4-fold more sensitive at the physiological concentration ranges than at concentrations >10 mM. Additionally, the channels have the propensity to dwell in the closed state at high concentrations of Sr2+, which is reflected in the time constant of the first latency distributions. It is concluded that the concentration of the permeant ion modulates the gating of hair cell Ca2+ channels. Finally, the closed state/s that is/are altered by high concentrations of Sr2+ may represent divalent ion-dependent inactivation of the L-type channel.

Sign in / Sign up

Export Citation Format

Share Document