scholarly journals Fundamental Gating Mechanism of Nicotinic Receptor Channel Revealed by Mutation Causing a Congenital Myasthenic Syndrome

2000 ◽  
Vol 116 (3) ◽  
pp. 449-462 ◽  
Author(s):  
Hai-Long Wang ◽  
Kinji Ohno ◽  
Margherita Milone ◽  
Joan M. Brengman ◽  
Amelia Evoli ◽  
...  
Keyword(s):  
Rate Constants ◽  
Single Channel ◽  
Gaussian Function ◽  
Congenital Myasthenic Syndrome ◽  
Markov Modeling ◽  
Amphipathic Helix ◽  
Wild Type ◽  
Gating Mechanism ◽  
Myasthenic Syndrome ◽  
Single Channel Currents

We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation εA411P in the amphipathic helix of the acetylcholine receptor (AChR) ε subunit. Myasthenic patients from three unrelated families are either homozygous for εA411P or are heterozygous and harbor a null mutation in the second ε allele, indicating that εA411P is recessive. We expressed human AChRs containing wild-type or A411P ε subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by εA411P. Prolines engineered into positions flanking residue 411 of the ε subunit greatly increase the range of activation kinetics similar to εA411P, whereas prolines engineered into positions equivalent to εA411 in β and δ subunits are without effect. Thus, the amphipathic helix of the ε subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.

A conserved cytoplasmic region of ROMK modulates pH sensitivity, conductance, and gating

1997 ◽  
Vol 273 (4) ◽  
pp. F516-F529 ◽  
Author(s):  
Han Choe ◽  
Hao Zhou ◽  
Lawrence G. Palmer ◽  
Henry Sackin
Keyword(s):  
Single Channel ◽  
Ph Sensitivity ◽  
Channel Noise ◽  
Wild Type ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Hydrophobic Region ◽  
K Secretion ◽  
Single Channel Currents

ROMK channels play a key role in overall K balance by controlling K secretion across the apical membrane of mammalian cortical collecting tubule. In contrast to the family of strong inward rectifiers (IRKs), ROMK channels are markedly sensitive to intracellular pH. Using Xenopus oocytes, we have confirmed this pH sensitivity at both the single-channel and whole cell level. Reduction of oocyte pH from 6.8 to 6.4 (using a permeant acetate buffer) reduced channel open probability from 0.76 ± 0.02 to near zero ( n = 8), without altering single-channel conductance. This was due to the appearance of a long-lived closed state at low internal pH. We have confirmed that a lysine residue (K61 on ROMK2; K80 on ROMK1), NH2 terminal to the first putative transmembrane segment (M1), is primarily responsible for conferring a steep pH sensitivity to ROMK (B. Fakler, J. Schultz, J. Yang, U. Schulte, U. Bråandle, H. P. Zenner, L. Y. Jan, and J. P. Ruppersberg. EMBO J. 15: 4093–4099, 1996). However, the apparent p K a of ROMK also depends on another residue in a highly conserved, mildly hydrophobic area: T51 on ROMK2 (T70 on ROMK1). Replacing this neutral threonine (T51) with a negatively charged glutamate shifted the apparent p K a for inward conductance from 6.5 ± 0.01 ( n = 8, wild type) to 7.0 ± 0.02 ( n = 5, T51E). On the other hand, replacing T51 with a positively charged lysine shifted the apparent p K a in the opposite direction, from 6.5 ± 0.01 ( n = 8, wild type) to 6.0 ± 0.02 ( n = 9, T51K). The opposite effects of the glutamate and lysine substitutions at position 51 (ROMK2) are consistent with a model in which T51 is physically close to K61 and alters either the local pH or the apparent p K a via an electrostatic mechanism. In addition to its effects on pH sensitivity, the mutation T51E also decreased single-channel conductance from 34.0 ± 1.0 pS ( n = 8, wild type) to 17.4 ± 1 pS ( n = 9, T51E), reversed the voltage gating of the channel, and significantly increased open-channel noise. These effects on single-channel currents suggest that the T51 residue, located in a mildly hydrophobic area of ROMK2, also interacts with the hydrophobic region of the permeation pathway.

scholarly journals Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR

2016 ◽  
Vol 148 (1) ◽  
pp. 43-63 ◽  
Author(s):  
Nuriya Mukhtasimova ◽  
Corrie J.B. daCosta ◽  
Steven M. Sine
Keyword(s):  
Rate Constants ◽  
Single Channel ◽  
Partial Agonist ◽  
Equilibrium Constants ◽  
Channel Gating ◽  
Kinetic Scheme ◽  
Step Response ◽  
Forward Rate ◽  
Gain Of Function ◽  
Single Channel Currents

The acetylcholine receptor (AChR) from vertebrate skeletal muscle initiates voluntary movement, and its kinetics of activation are crucial for maintaining the safety margin for neuromuscular transmission. Furthermore, the kinetic mechanism of the muscle AChR serves as an archetype for understanding activation mechanisms of related receptors from the Cys-loop superfamily. Here we record currents through single muscle AChR channels with improved temporal resolution approaching half an order of magnitude over our previous best. A range of concentrations of full and partial agonists are used to elicit currents from human wild-type and gain-of-function mutant AChRs. For each agonist–receptor combination, rate constants are estimated from maximum likelihood analysis using a kinetic scheme comprised of agonist binding, priming, and channel gating steps. The kinetic scheme and rate constants are tested by stochastic simulation, followed by incorporation of the experimental step response, sampling rate, background noise, and filter bandwidth. Analyses of the simulated data confirm all rate constants except those for channel gating, which are overestimated because of the established effect of noise on the briefest dwell times. Estimates of the gating rate constants were obtained through iterative simulation followed by kinetic fitting. The results reveal that the agonist association rate constants are independent of agonist occupancy but depend on receptor state, whereas those for agonist dissociation depend on occupancy but not on state. The priming rate and equilibrium constants increase with successive agonist occupancy, and for a full agonist, the forward rate constant increases more than the equilibrium constant; for a partial agonist, the forward rate and equilibrium constants increase equally. The gating rate and equilibrium constants also increase with successive agonist occupancy, but unlike priming, the equilibrium constants increase more than the forward rate constants. As observed for a full and a partial agonist, the gain-of-function mutation affects the relationship between rate and equilibrium constants for priming but not for channel gating. Thus, resolving brief single channel currents distinguishes priming from gating steps and reveals how the corresponding rate and equilibrium constants depend on agonist occupancy.

scholarly journals Stoichiometry of Recombinant N-Methyl-d-Aspartate Receptor Channels Inferred from Single-channel Current Patterns

1997 ◽  
Vol 110 (5) ◽  
pp. 485-502 ◽  
Author(s):  
Louis S. Premkumar ◽  
Anthony Auerbach
Keyword(s):  
Single Channel ◽  
Wild Type ◽  
Current Pattern ◽  
Channel Current ◽  
Single Channel Current ◽  
Aspartate Receptor ◽  
Single Channel Currents ◽  
Nr2 Subunits ◽  
Channel Currents ◽  
Straightforward Interpretation

Single-channel currents were recorded from mouse NR1-NR2B (ζ-ε2) receptors containing mixtures of wild-type and mutant subunits expressed in Xenopus oocytes. Mutant subunits had an asparagine-to-glutamine (N-to-Q) mutation at the N0 site of the M2 segment (NR1:598, NR2B:589). Receptors with pure N or Q NR1 and NR2 subunits generated single-channel currents with distinctive current patterns. Based on main and sublevel amplitudes, occupancy probabilities, and lifetimes, four patterns of current were identified, corresponding to receptors with the following subunit compositions (NR1/NR2): N/N, N/Q, Q/N, and Q/Q. Only one current pattern was apparent for each composition. When a mixture of N and Q NR2 subunits was coexpressed with pure mutant NR1 subunits, three single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as Q/N receptors. The third, novel pattern presumably arose from hybrid receptors having both N and Q NR2 subunits. When a mixture of N and Q NR1 subunits was coexpressed with pure mutant NR2 subunits, six single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as N/Q receptors. The four novel patterns presumably arose from hybrid receptors having both N and Q NR1 subunits. The relative frequency of NR1 hybrid receptor current patterns depended on the relative amounts of Q and N subunits that were injected into the oocytes. The number of hybrid receptor patterns suggests that there are two NR2 subunits per receptor and is consistent with either three or five NR1 subunits per receptor, depending on whether or not the order of mutant and wild-type subunits influences the current pattern. When considered in relation to other studies, the most straightforward interpretation of the results is that N-methyl-d-aspartate receptors are pentamers composed of three NR1 and two NR2 subunits.

scholarly journals Single-Channel Characteristics of Wild-Type IKs Channels and Channels formed with Two MinK Mutants that Cause Long QT Syndrome

1998 ◽  
Vol 112 (6) ◽  
pp. 651-663 ◽  
Author(s):  
Federico Sesti ◽  
Steve A.N. Goldstein
Keyword(s):  
Long Qt Syndrome ◽  
Single Channel ◽  
Xenopus Laevis Oocytes ◽  
Wild Type ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Long Qt ◽  
Conduction Pathway ◽  
Qt Syndrome ◽  
Single Channel Currents

IKs channels are voltage dependent and K+ selective. They influence cardiac action potential duration through their contribution to myocyte repolarization. Assembled from minK and KvLQT1 subunits, IKs channels are notable for a heteromeric ion conduction pathway in which both subunit types contribute to pore formation. This study was undertaken to assess the effects of minK on pore function. We first characterized the properties of wild-type human IKs channels and channels formed only of KvLQT1 subunits. Channels were expressed in Xenopus laevis oocytes or Chinese hamster ovary cells and currents recorded in excised membrane patches or whole-cell mode. Unitary conductance estimates were dependent on bandwidth due to rapid channel “flicker.” At 25 kHz in symmetrical 100-mM KCl, the single-channel conductance of IKs channels was ∼16 pS (corresponding to ∼0.8 pA at 50 mV) as judged by noise-variance analysis; this was fourfold greater than the estimated conductance of homomeric KvLQT1 channels. Mutant IKs channels formed with D76N and S74L minK subunits are associated with long QT syndrome. When compared with wild type, mutant channels showed lower unitary currents and diminished open probabilities with only minor changes in ion permeabilities. Apparently, the mutations altered single-channel currents at a site in the pore distinct from the ion selectivity apparatus. Patients carrying these mutant minK genes are expected to manifest decreased K+ flux through IKs channels due to lowered single-channel conductance and altered gating.

scholarly journals The Extracellular Linker of Muscle Acetylcholine Receptor Channels Is a Gating Control Element

2000 ◽  
Vol 116 (3) ◽  
pp. 327-340 ◽  
Author(s):  
Claudio Grosman ◽  
Frank N. Salamone ◽  
Steven M. Sine ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptors ◽  
Single Channel ◽  
Channel Gating ◽  
Congenital Myasthenic Syndrome ◽  
Control Element ◽  
Dependent Manner ◽  
Α Subunit ◽  
Transmembrane Segments ◽  
Channel Opening ◽  
Myasthenic Syndrome

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2–M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the α subunit (αS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the αS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an ∼92-fold increased gating equilibrium constant, which is consistent with an ∼10-fold decreased EC50 in the presence of ACh. With choline, this mutation accelerates channel opening ∼28-fold, slows channel closing ∼3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, αS269I acetylcholine receptors open at a rate of ∼1.4 × 106 s−1 and close at a rate of ∼760 s−1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of ∼140 s−1. Ile mutations at positions flanking αS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the α subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the αS269I mutation, Ile mutations at equivalent positions of the β, ε, and δ subunits do not affect apparent open-channel lifetimes. However, in β and ε, shifting the mutation one residue to the NH2-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2–M3L of the α subunit moves before the corresponding linkers of the β and ε subunits.

scholarly journals Decremental Response to High-Frequency Trains of Acetylcholine Pulses but Unaltered Fractional Ca2+ Currents in a Panel of “Slow-Channel Syndrome” Nicotinic Receptor Mutants

2009 ◽  
Vol 133 (2) ◽  
pp. 151-169 ◽  
Author(s):  
Sergio Elenes ◽  
Michael Decker ◽  
Gisela D. Cymes ◽  
Claudio Grosman
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Congenital Myasthenic Syndrome ◽  
Physiological Importance ◽  
Naturally Occurring ◽  
Slow Channel ◽  
Myasthenic Syndrome ◽  
Extracellular Side ◽  
Mutant Receptors ◽  
Kinetics Of

The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular junction caused by gain-of-function mutations to the muscle nicotinic acetylcholine (ACh) receptor (AChR). Although it is clear that the slower deactivation time course of the ACh-elicited currents plays a central role in the etiology of this disease, it has been suggested that other abnormal properties of these mutant receptors may also be critical in this respect. We characterized the kinetics of a panel of five SCCMS AChRs (αS269I, βV266M, ɛL221F, ɛT264P, and ɛL269F) at the ensemble level in rapidly perfused outside-out patches. We found that, for all of these mutants, the peak-current amplitude decreases along trains of nearly saturating ACh pulses delivered at physiologically relevant frequencies in a manner that is consistent with enhanced entry into desensitization during the prolonged deactivation phase. This suggests that the increasingly reduced availability of activatable AChRs upon repetitive stimulation may well contribute to the fatigability and weakness of skeletal muscle that characterize this disease. Also, these results emphasize the importance of explicitly accounting for entry into desensitization as one of the pathways for burst termination, if meaningful mechanistic insight is to be inferred from the study of the effect of these naturally occurring mutations on channel function. Applying a novel single-channel–based approach to estimate the contribution of Ca2+ to the total cation currents, we also found that none of these mutants affects the Ca2+-conduction properties of the AChR to an extent that seems to be of physiological importance. Our estimate of the Ca2+-carried component of the total (inward) conductance of wild-type and SCCMS AChRs in the presence of 150 mM Na+, 1.8 mM Ca2+, and 1.7 mM Mg2+ on the extracellular side of cell-attached patches turned out be in the 5.0–9.4 pS range, representing a fractional Ca2+ current of ∼14%, on average. Remarkably, these values are nearly identical to those we estimated for the NR1-NR2A N-methyl-d-aspartate receptor (NMDAR), which has generally been considered to be the main neurotransmitter-gated pathway of Ca2+ entry into the cell. Our estimate of the rat NMDAR Ca2+ conductance (using the same single-channel approach as for the AChR but in the nominal absence of extracellular Mg2+) was 7.9 pS, corresponding to a fractional Ca2+ current of 13%.

scholarly journals Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

1997 ◽  
Vol 109 (6) ◽  
pp. 757-766 ◽  
Author(s):  
Hai-Long Wang ◽  
Anthony Auerbach ◽  
Nina Bren ◽  
Kinji Ohno ◽  
Andrew G. Engel ◽  
...  
Keyword(s):  
Kinetic Analysis ◽  
Acetylcholine Receptor ◽  
Rate Constants ◽  
Single Channel ◽  
Receptor Activation ◽  
Congenital Myasthenic Syndrome ◽  
Α Subunit ◽  
Apparent Affinity ◽  
Amino Terminal ◽  
Channel Opening

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.

scholarly journals Rate theory calculation of gramicidin single-channel currents using NMR-derived rate constants.

1980 ◽  
Vol 77 (4) ◽  
pp. 2028-2032 ◽  
Author(s):  
D. W. Urry ◽  
C. M. Venkatachalam ◽  
A. Spisni ◽  
P. Lauger ◽  
M. A. Khaled
Keyword(s):  
Rate Constants ◽  
Single Channel ◽  
Rate Theory ◽  
Single Channel Currents ◽  
Theory Calculation ◽  
Channel Currents

The malonyl gramicidin channel: NMR-derived rate constants and comparison of calculated and experimental single-channel currents

1980 ◽  
Vol 55 (1) ◽  
pp. 29-51 ◽  
Author(s):  
D. W. Urry ◽  
C. M. Venkatachalam ◽  
A. Spisni ◽  
R. J. Bradley ◽  
T. L. Trapane ◽  
...  
Keyword(s):  
Rate Constants ◽  
Single Channel ◽  
Gramicidin Channel ◽  
Single Channel Currents ◽  
Channel Currents

scholarly journals Naturally Occurring Mutations at the Acetylcholine Receptor Binding Site Independently Alter ACh Binding and Channel Gating

2002 ◽  
Vol 120 (4) ◽  
pp. 483-496 ◽  
Author(s):  
Steven M. Sine ◽  
Xing-Ming Shen ◽  
Hai-Long Wang ◽  
Kinji Ohno ◽  
Won-Yong Lee ◽  
...  
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Rate Constants ◽  
Structural Model ◽  
Single Channel ◽  
Channel Gating ◽  
Congenital Myasthenic Syndrome ◽  
Protein Chain ◽  
Α Subunit ◽  
Closed State

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, εN182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant αδ site. Studies of the analogous mutation in the δ subunit, δN187Y, disclose rate constants for ACh occupancy of the nonmutant αε site. The second CMS mutation, εD175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. εD175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, εN182Y localizes to the interface with the α subunit, and εD175 to the entrance of the ACh binding cavity. Both εN182Y and εD175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring εN182 and εD175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.

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