Evidence that extracellular anions interact with a site outside the CFTR chloride channel pore to modify channel properties

2009 ◽  
Vol 87 (5) ◽  
pp. 387-395 ◽  
Author(s):  
Jing-Jun Zhou ◽  
Paul Linsdell
Keyword(s):  
Long Range ◽  
Single Channel ◽  
Site Directed Mutagenesis ◽  
Extracellular Loop ◽  
Patch Clamp Recording ◽  
Pore Region ◽  
Channel Pore ◽  
Single Channel Currents ◽  
A Site ◽  
Channel Properties

Extracellular anions enter into the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel, interacting with binding sites on the pore walls and with other anions inside the pore. There is increasing evidence that extracellular anions may also interact with sites away from the channel pore to influence channel properties. We have used site-directed mutagenesis and patch-clamp recording to identify residues that influence interactions with external anions. Anion interactions were assessed by the ability of extracellular Pt(NO2)42– ions to weaken the pore-blocking effect of intracellular Pt(NO2)42– ions, a long-range ion–ion interaction that does not appear to reflect ion interactions inside the pore. We found that mutations that remove positive charges in the 4th extracellular loop of CFTR (K892Q and R899Q) significantly alter the interaction between extracellular and intracellular Pt(NO2)42– ions. These mutations do not affect unitary Cl– conductance or block of single-channel currents by extracellular Pt(NO2)42– ions, however, suggesting that the mutated residues are not in the channel pore region. These results suggest that extracellular anions can regulate CFTR pore properties by binding to a site outside the pore region, probably by a long-range conformational change. Our findings also point to a novel function of the long 4th extracellular loop of the CFTR protein in sensing and (or) responding to anions in the extracellular solution.

scholarly journals The Pore Architecture of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Revealed by Co-Mutation in Pore-Forming Transmembrane Regions

2016 ◽  
pp. 505-515
Author(s):  
F. QIAN ◽  
L. LIU ◽  
Z. LIU ◽  
C. LU
Keyword(s):  
Cystic Fibrosis ◽  
Single Channel ◽  
Transmembrane Conductance Regulator ◽  
Patch Clamp Recording ◽  
Transmembrane Conductance ◽  
Channel Pore ◽  
Selective Filter ◽  
Transmembrane Regions ◽  
Cystic Fibrosis Transmembrane ◽  
Insight Into

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel contains 12 transmembrane (TM) regions that are presumed to form the channel pore. However, there is no direct evidence clearly illustrating the involvement of these transmembrane regions in the actual CFTR pore structure. To obtain insight into the architecture of the CFTR channel pore, we used patch clamp recording techniques and a strategy of co-mutagenesis of two potential pore-forming transmembrane regions (TM1 and TM6) to investigate the collaboration of these two TM regions. We performed a range of specific functional assays comparing the single channel conductance, anion binding, and anion selectivity properties of the co-mutated CFTR variants, and the results indicated that TM1 and TM6 play vital roles in forming the channel pore and, thus, determine the functional properties of the channel. Furthermore, we provided functional evidence that the amino acid threonine (T338) in TM6 has synergic effects with lysine (K95) in TM1. Therefore, we propose that these two residues have functional collaboration in the CFTR channel pore and may collectively form a selective filter.

Recording of single γ-aminobutyrate- and acetylcholine-activated receptor channels translated by exogenous mRNA in Xenopus oocytes

1983 ◽  
Vol 218 (1213) ◽  
pp. 481-484 ◽  
Keyword(s):  
High Resolution ◽  
Patch Clamp ◽  
Xenopus Oocytes ◽  
Optic Lobe ◽  
Single Channel ◽  
Patch Clamp Recording ◽  
Chick Optic Lobe ◽  
Single Channel Currents ◽  
Channel Currents

High resolution (‘giga-seal’) patch clamp recording in Xenopus oocytes was used to measure single channel currents from ACh- and GABA-activated receptors. The proteins that make up these receptors had been translated from mRNA derived from, respectively, denervated cat muscle and chick optic lobe.

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.

Sodium current and membrane potential in EDL muscle fibers from normal and dystrophic (mdx) mice

1991 ◽  
Vol 261 (4) ◽  
pp. C718-C725 ◽  
Author(s):  
C. Mathes ◽  
F. Bezanilla ◽  
R. E. Weiss
Keyword(s):  
Membrane Potential ◽  
Sodium Channels ◽  
Single Channel ◽  
Muscle Fibers ◽  
Mdx Mice ◽  
Apparent Difference ◽  
Sodium Currents ◽  
Edl Muscle ◽  
Single Channel Currents ◽  
Channel Properties

The macroscopic and single-channel properties of sodium currents and membrane potential were studied in intact extensor digitorum longus (EDL) muscle fibers from mdx (C57BL/10ScSn-mdx) and normal (C57BL/10SnJ) mice. The voltage dependence of activation and inactivation were determined and the associated gating charges were calculated to determine if the lack of dystrophin associated with the mdx condition has any influence on sodium channels either directly or by effects on the membrane environment of the channel. Sodium currents were recorded from cell-attached patches on EDL muscle fibers isolated by collagenase treatment and manual dissection. Both macroscopic and single-channel currents were studied. We found no apparent difference in the sodium channel properties from the two types of muscle. In addition, microelectrode measurements in both mdx and normal muscle fibers indicated similar resting membrane potentials (Vm around -95 mV), which suggests that the normal behavior of sodium channels in the muscle sarcolemma is unaffected by the X-linked gene defect.

Surfing the Conformational Wave of Acetylcholine Receptor Channel Gating

2001 ◽  
Vol 7 (S2) ◽  
pp. 24-25
Author(s):  
Gisela Cymes ◽  
Claudio Grosman ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptor ◽  
Rate Constant ◽  
Single Molecule ◽  
Single Channel ◽  
Site Directed Mutagenesis ◽  
Kinetic Measurements ◽  
Ion Conducting ◽  
Single Channel Currents ◽  
Acetylcholine Receptor Channel ◽  
Receptor Channel

The muscle nicotinic acetylcholine receptor channel (AChR) is a cylindrical allosteric membrane protein (∼120 x 60 Å Fig. 1) that adopts alternative quaternary conformations (“open” and “closed”) with different functional properties (ion-conducting and ion-impermeable, respectively). We have characterized, residue-by-residue, the dynamics of the conformational change associated with gating using the framework of linear free energy relationships (LFER). The sequence of molecular events that underlies the closed-to-open gating transition was inferred from kinetic measurements of the receptor at the single molecule level.Specific regions of the AChR were perturbed using site-directed mutagenesis, changes in the membrane potential, or different agonists. Single-channel currents were recorded from cell-attached patches (Fig. 2). For the gain-of-function mutations, choline was used as the agonist because of its low efficacy. The opening rate constant was determined at a saturating concentration of agonist (for choline, 20 mM) in order to isolate gating from binding steps. to avoid bias introduced by fast channel blockade, the closing rate constant was measured at a low concentration (for choline, 200 μM). The diliganded channel opening (β) and closing (α) rate constants were estimated using the QuB suite of kinetic analysis programs. in general, a plot of the log rate constant vs. log equilibrium constant was linear.

Multiple Mechanisms of Ketamine Blockade of N-methyl-D-aspartate Receptors 

1997 ◽  
Vol 86 (4) ◽  
pp. 903-917 ◽  
Author(s):  
Beverley A. Orser ◽  
Peter S. Pennefather ◽  
John F. MacDonald
Keyword(s):  
Hippocampal Neurons ◽  
Open Channel ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Open Time ◽  
Channel Opening ◽  
Single Channel Currents ◽  
A Site ◽  
Change In Mean

Background The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is blocked by ketamine, and this action likely contributes to ketamine's anesthetic and analgesic properties. Previous studies suggest that ketamine occludes the open channel by binding to a site located within the channel pore. This hypothesis was examined by investigating the effects of ketamine on single-channel currents from NMDA receptors. Methods The cell-attached and outside-out configurations of the patch clamp technique were used to study NMDA-activated currents recorded from cultured mouse hippocampal neurons. Results In cell-attached patches, NMDA evoked currents that had an apparent mean open time (tau o) of 3.26 ms. The probability of at least one channel being open (Po') was 0.058. The addition of ketamine (0.1 microM or 1 microM) to the pipette solution decreased Po' to 53% and 24% of control values, respectively. At 1 microM ketamine, this reduction was due to a decrease in both the frequency of channel opening and the mean open time (44% and 68% of control values, respectively). Ketamine did not influence channel conductance and no new components were required to fit the open- or closed-duration distributions. Ketamine (50 microM), applied outside the recording pipette, reduced the opening frequency of channels recorded in the cell attached configuration. This observation suggests that ketamine gained access to a binding site by diffusing across the hydrophobic cell membrane. In outside-out patches, ketamine potency was lower than that observed in cell-attached patches: 1 microM and 10 microM ketamine reduced Po' to 63% and 34% of control values, respectively, and this reduction was due primarily to a decrease in the frequency of channel opening with little change in mean open time. Conclusions These observations are consistent with a model whereby ketamine inhibits the NMDA receptor by two distinct mechanisms: (1) Ketamine blocks the open channel and thereby reduces channel mean open time, and (2) ketamine decreases the frequency of channel opening by an allosteric mechanism.

Similar properties of cGMP-activated channels between cones and rods in the carp retina

1991 ◽  
Vol 6 (6) ◽  
pp. 563-568 ◽  
Author(s):  
Shu-Ichi Watanabe ◽  
Motohiki Murakami
Keyword(s):  
Animal Species ◽  
Single Channel ◽  
Tiger Salamander ◽  
Membrane Hyperpolarization ◽  
Rods And Cones ◽  
Low Concentrations ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Inside Out ◽  
Channel Properties

AbstractUsing patch-clamp techniques, properties of cGMP-activated channel were studied at a single-channel level in order to examine (1) whether any differences are recognized between the cGMP-activated channels of rods and cones in the same animal species, and (2) whether the channel properties of the same photoreceptor class differ in different animal species. Experiments were performed on inside-out membrane patches excised from outer segments of rods and morphological subtypes of cones in the carp retina. Single-channel activities could be recorded when the patches were perfused with low concentrations of cGMP (<10 μM). Throughout five morphological subtypes of cones and rod, single-channel currents showed no significant rectification at membrane hyperpolarization in a low divalent cation solution, and single-channel conductances were almost the same: 13.8 ± 0.2 pS (mean ± s.e.m., n = 23) in cones and 12.7 ± 0.8 pS (n = 3) in rods. These values were significantly smaller than that reported in catfish cones (about 50 pS), and that in rods of the toad and the tiger salamander (about 25 pS). In rods and all subtypes of cones of the carp, open durations of cGMP activated channels were brief. In addition, kinetic parameters of channel openings and closings showed no differences throughout all subtypes of cones and rod.

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.

Whole cell and single channel properties of a new GABA receptor transiently expressed in the Hippocampus

1995 ◽  
Vol 73 (2) ◽  
pp. 902-906 ◽  
Author(s):  
M. Martina ◽  
F. Strata ◽  
E. Cherubini
Keyword(s):  
Postnatal Development ◽  
Hippocampal Neurons ◽  
Single Channel ◽  
Gamma Aminobutyric Acid ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Slow Kinetics ◽  
Inward Currents ◽  
Single Channel Currents ◽  
Channel Properties

1. The patch-clamp technique was used to characterize, in acutely dissociated CA3 rat hippocampal neurons, the whole cell and single channel properties of a novel response to gamma-aminobutyric acid (GABA) present only during a restricted period of postnatal development. 2. At postnatal days 0-10 (P0-P10), both GABA (100 microM) and isoguvacine (50 microM) evoked at a holding potential of -50 mV, in symmetrical chloride solution, whole cell inward currents. Bicuculline blocked the response to isoguvacine but only reduced the response to GABA (from 512 +/- 137 pA to 60 +/- 13 pA, mean +/- SE). After P12, bicuculline abolished the response to GABA. 3. The bicuculline-insensitive GABA currents were Cl- mediated and antagonized by picrotoxin. The desensitization rate was slower than the conventional bicuculline-sensitive response. The peak to plateau ratio induced by 0.1 or 1 mM of GABA shifted from 4.6 +/- 0.4 and 17.7 +/- 2.6 to 1.5 +/- 0.1 and 3.1 +/- 0.5 in the absence or in the presence of bicuculline, respectively. The recovery from desensitization was significantly faster for the bicuculline-insensitive responses. 4. In excised outside-out patches, GABA (20 microM) activated, in the presence of bicuculline (100 microM), single channel currents having conductances of 14, 22, and 31 pS. These values were similar to those obtained in the same preparation, in the absence of bicuculline. 5. These findings suggest that this new receptor type, which mediates bicuculline-insensitive responses with slow kinetics, may potentiate the depolarizing action of GABA during a critical period of postnatal development and therefore play a crucial role in synaptogenesis.

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