scholarly journals Functional Geometry of the Permeation Pathway of Ca2+-activated Cl−Channels Inferred from Analysis of Voltage-dependent Block

2001 ◽  
Vol 276 (21) ◽  
pp. 18423-18429 ◽  
Author(s):  
Zhiqiang Qu ◽  
H. Criss Hartzell
Keyword(s):  
Voltage Dependent ◽  
Cl Channels ◽  
Functional Geometry

scholarly journals A chloride channel from lobster walking leg nerves. Characterization of single-channel properties in planar bilayers.

1990 ◽  
Vol 96 (4) ◽  
pp. 707-733 ◽  
Author(s):  
G L Lukács ◽  
E Moczydlowski
Keyword(s):  
Single Channel ◽  
Disulfonic Acid ◽  
Ph Change ◽  
Planar Bilayers ◽  
Voltage Range ◽  
Cl Channel ◽  
Voltage Dependent ◽  
Cl Channels ◽  
Transport Inhibitors ◽  
Small Conductance

A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, single Cl- channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 +/- 0.8 pS at positive voltages and 15.1 +/- 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na+ compared with Cl- and displays the following sequence of anion permeability relative to Cl- as measured under near bi-ionic conditions: I- (2.7) greater than NO3- (1.8) greater than Br- (1.5) greater than Cl- (1.0) greater than CH3CO2- (0.18) greater than HCO3- (0.10) greater than gluconate (0.06) greater than F- (0.05). The unitary conductance saturates with increasing Cl- concentration in a Michaelis-Menten fashion with a Km of 100 mM and gamma max = 33 pS at positive voltage. The I-V curve is similar in 10 mM Tris or 10 mM HEPES buffer, but substitution of 100 mM NaCl with 100 mM tetraethylammonium chloride on the cis side results in increased rectification with a 40% reduction in current at negative voltages. The gating of the channel is weakly voltage dependent with an open-state probability of 0.23 at -75 mV and 0.64 at +75 mV. Channel gating is sensitive to cis pH with an increased opening probability observed for a pH change of 7.4 to 11 and nearly complete inhibition for a pH change of 7.4 to 6.0. The lobster Cl- channel is reversibly blocked by the anion transport inhibitors, SITS (4-acetamido, 4'-isothiocyanostilbene-2,2'-disulfonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid). Many of these characteristics are similar to those previously described for small conductance Cl- channels in various vertebrate cells, including epithelia. These functional comparisons suggest that this invertebrate Cl- channel is an evolutionary prototype of a widely distributed class of small conductance anion channels.

scholarly journals A Potassium-Selective Current Affected by Micromolar Concentrations of Anion Transport Inhibitors

2018 ◽  
Vol 45 (3) ◽  
pp. 867-882
Author(s):  
Roberta Costa ◽  
Davide Antonio Civello ◽  
Emanuele Bernardinelli ◽  
Simone Vanoni ◽  
Michaela Zopf ◽  
...  
Keyword(s):  
Niflumic Acid ◽  
Disulfonic Acid ◽  
Hek 293 ◽  
K Channels ◽  
Inflammatory Drug ◽  
Physiological Phenomenon ◽  
Voltage Dependent ◽  
Cl Channels ◽  
K Current ◽  
Anti Inflammatory

Background/Aims: In the human genome, more than 400 genes encode ion channels, which are ubiquitously expressed and often coexist and participate in almost all physiological processes. Therefore, ion channel blockers represent fundamental tools in discriminating the contribution of individual channel types to a physiological phenomenon. However, unspecific effects of these compounds may represent a confounding factor. Three commonly used chloride channel inhibitors, i.e. 4,4′-diisothiocyano-2,2′-stilbene-disulfonic acid (DIDS), 5-nitro-2-[(3-phenylpropyl) amino]benzoic acid (NPPB) and the anti-inflammatory drug niflumic acid were tested to identify the lowest concentration effective on Cl- channels and ineffective on K+ channels. Methods: The activity of the above mentioned compounds was tested by whole cell patch-clamp on the swelling-activated Cl- current ICl,swell and on the endogenous voltage-dependent, outwardly rectifying K+ selective current in human kidney cell lines (HEK 293/HEK 293 Phoenix). Results: Micromolar (1-10 µM) concentrations of DIDS and NPPB could not discriminate between the Cl- and K+ selective currents. Specifically, 1 µM DIDS only affected the K+ current and 10 µM NPPB equally affected the Cl- and K+ currents. Only relatively high (0.1-1 mM) concentrations of DIDS and prolonged (5 minutes) exposure to 0.1-1 mM NPPB preferentially suppressed the Cl- current. Niflumic acid preferentially inhibited the Cl- current, but also significantly affected the K+ current. The endogenous voltage-dependent, outwardly rectifying K+ selective current in HEK 293/HEK 293 Phoenix cells was shown to arise from the Kv 3.1 channel, which is extensively expressed in brain and is involved in neurological diseases. Conclusion: The results of the present study underscore that sensitivity of a given physiological phenomenon to the Cl- channel inhibitors NPPB, DIDS and niflumic acid may actually arise from an inhibition of Cl- channels but can also result from an inhibition of voltage-dependent K+ channels, including the Kv 3.1 channel. The use of niflumic acid as anti-inflammatory drug in patients with concomitant Kv 3.1 dysfunction may result contraindicated.

Diverse modulations of chloride channels in renal proximal tubules

1994 ◽  
Vol 267 (5) ◽  
pp. F716-F724 ◽  
Author(s):  
N. Darvish ◽  
J. Winaver ◽  
D. Dagan
Keyword(s):  
Chloride Channels ◽  
Disulfonic Acid ◽  
Channel Activity ◽  
Cation Permeability ◽  
Voltage Dependent ◽  
Selective Channel ◽  
Cl Channels ◽  
The Rich ◽  
Apical Membranes ◽  
Inside Out

Cl- selective channels were detected and characterized in apical membranes of cultured rat renal proximal convoluted tubule cells (PCT) using patch-clamping methods. Subpopulations of Cl- channels modulated by cyclic nucleotides, Ca2+, or voltage were identified. Two different 30-pS, voltage-independent, Cl- channels modulated by adenosine 3',5'-cyclic monophosphate (cAMP) or Ca2+ were seen most frequently. The cAMP-dependent channel was activated by membrane-permeable analogues of cAMP, dibutyryl-cAMP or 8-bromo-cAMP. Catalytic subunit of protein kinase A (PKA) applied to detached inside-out patches, activated the channel as well, suggesting activation via phosphorylation. Channel activity was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, by 4,4-dinitrostilbene-2,2-disulfonic acid, and by SCN-. Permeability sequence for different halides was Cl- > I > F with a Cl(-)-to-cation permeability ratio (PCl/Pcation) of 7:1. The Ca(2+)-sensitive channel was not activated by cAMP nor by PKA. A third anionic selective channel encountered infrequently is voltage dependent and has a unitary conductance of 145 pS, with a PCl/Pcation value of 9:1. This diversity of Cl- channels may underlie the rich repertoire of physiological functions attributed to Cl- channels.

Double-barreled chloride channels of collecting duct basolateral membrane

1990 ◽  
Vol 259 (1) ◽  
pp. F46-F52 ◽  
Author(s):  
S. C. Sansom ◽  
B. Q. La ◽  
S. L. Carosi
Keyword(s):  
Chloride Channels ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Electric Organ ◽  
Cortical Collecting Duct ◽  
Cl Channel ◽  
Voltage Dependent ◽  
Cl Channels ◽  
Excised Patches ◽  
High Concentrations

Microelectrode studies have shown that the basolateral membrane of the principal cells (PC) of the rabbit cortical collecting duct (CCD) contains Cl(-)-conductive pathways. To determine the properties of single Cl- channels we prepared the basolateral membrane for patch clamping by incubating the CCD in collagenase and/or tearing the basement membrane with a fine needle. When high concentrations of collagenase were used, only a small nonselective channel was observed. In low concentrations or the absence of collagenase, however, we identified a Cl- channel (g46) in both cell-attached and excised patches. The Cl- channel gated rapidly between three equally spaced substates of 0 (S0), 23 (S1), and 46 pS (S2) and slowly between states C (inactive) and S0. The conductance of each substate was not voltage dependent between pipette potentials from -60 to +60 mV (cell attached). The probability that the channel gated from C to S0 increased with hyperpolarizing potentials, but the probability that g46 was in substate S0 increased with depolarizing patch potentials. This channel was similar to that described by Miller for the nonexcitable membrane of the electric organ of Torpedo californica. Because g46 was the most frequently observed basolateral membrane channel and was voltage dependent at physiological potentials, it is probably the channel responsible for the dominant Cl- conductance of PC.

scholarly journals Secretory activation of basolateral membrane Cl−channels in guinea pig distal colonic crypts

2003 ◽  
Vol 284 (4) ◽  
pp. C918-C933 ◽  
Author(s):  
Yingjun Li ◽  
Susan Troutman Halm ◽  
Dan R. Halm
Keyword(s):  
Guinea Pig ◽  
Relative Permeability ◽  
Single Channel ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Resting Membrane Potential ◽  
Colonic Crypts ◽  
Voltage Dependent ◽  
Cl Channels ◽  
Excised Patches

Cell-attached recordings revealed Cl− channel activity in basolateral membrane of guinea pig distal colonic crypts isolated from basement membrane. Outwardly rectified currents (gpClor) were apparent with a single-channel conductance (γ) of 29 pS at resting membrane electrical potential; another outward rectifier with γ of 24 pS was also observed (∼25% of gpClor). At a holding potential of −80 mV γ was 18 pS for bothgpClor currents, and at +80 mV γ was 67 and 40 pS, respectively. Identity as Cl− channels was confirmed in excised patches by changing bath ion composition. From reversal potentials, relative permeability of K+ over Cl− ( P K/ P Cl) was 0.07 ± 0.03, with relative permeability of Na+over Cl−( P Na/ P Cl) = 0.08 ± 0.04. A second type of Cl− channel was seen with linear current-voltage ( I-V) relations (gpClL), having subtypes with γ of 21, 13, and 8 pS. Epinephrine or forskolin increased the number of opengpClor and gpClL. Open probabilities ( P o) ofgpClor, gpClL21, andgpClL13 were voltage dependent in cell-attached patches, higher at more positive potentials. Kinetics ofgpClor were more rapid with epinephrine activation than with forskolin activation. Epinephrine increased P o at the resting membrane potential forgpClL13. Secretagogue activation of these Cl− channels may contribute to stimulation of electrogenic K+ secretion across colonic epithelium by increasing basolateral membrane Cl− conductance that permits Cl− exit after uptake via Na+-K+-2Cl− cotransport.

Volume-activated chloride channels in HL-60 cells: potent inhibition by an oxonol dye

1995 ◽  
Vol 269 (4) ◽  
pp. C1063-C1072 ◽  
Author(s):  
J. Arreola ◽  
K. R. Hallows ◽  
P. A. Knauf
Keyword(s):  
Chloride Channels ◽  
Regulatory Volume Decrease ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Independent Manner ◽  
Low Concentrations ◽  
Potent Inhibition ◽  
Voltage Dependent ◽  
Cl Channels

When swollen in hypotonic media, HL-60 cells exhibit a regulatory volume decrease (RVD) response as a result of net losses of K+ and Cl-. This is primarily caused by a dramatic increase in Cl- permeability, which may reflect the opening of volume-sensitive channels (11). To test this hypothesis, we measured volume-activated Cl- currents in HL-60 cells using the patch-clamp technique. The whole cell Cl- conductance (in nS/pF at 100 mV) increased from 0.09 +/- 0.06 to 1.15 +/- 0.19 to 1.64 +/- 0.40 as the tonicity (in mosmol/kgH2O) of the external medium was decreased from 334 to 263 to 164, respectively. Cl- currents showed no significant inactivation during 800-ms pulses. Current-voltage curves exhibited outward rectification and were identical at holding potentials of 0 or -50 mV, suggesting that the gating of the channels is voltage independent. The selectivity sequence, based on permeability ratios (PX/PCl) calculated from the shifts of the reversal potentials, was SCN- > I- approximately NO3- > Br- > Cl- >> gluconate. 4-Acetamido-4'- isothiocyanostilbene-2,2'-disulfonic acid (SITS; 0.5 mM) inhibits HL-60 Cl- channels in a voltage-dependent manner, with approximately 10-fold increased affinity at potentials greater than +40 mV. Voltage-dependent blockade by SITS indicates that the binding site is located near the outside, where it senses 20% of the membrane potential. These Cl- channels were also inhibited in a voltage-independent manner by the oxonol dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol [diBA-(5)-C4] with a concentration that gives half inhibition (IC50) of 1.8 microM at room temperature. A similar apparent IC50 value (1.2 microM) was observed for net 36Cl- efflux into a Cl(-)-free hypotonic medium at 21 degrees C. It seems likely, therefore, that the volume-activated Cl- channels are responsible for the net Cl- efflux during RVD. These Cl- channels have properties similar to the “mini-Cl-” channels described in lymphocytes and neutrophils and are strongly inhibited by low concentrations of diBA-(5)-C4.

scholarly journals Generation and Role of Oscillatory Contractions in Mouse Airway Smooth Muscle

2018 ◽  
Vol 47 (4) ◽  
pp. 1546-1555 ◽  
Author(s):  
Hao Xu ◽  
Ping Zhao ◽  
Wen-Jing Zhang ◽  
Jun-Ying Qiu ◽  
Li Tan ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Imaging Technique ◽  
Tetraethylammonium Chloride ◽  
Inositol 1,4,5 Trisphosphate ◽  
Acetylcholine Chloride ◽  
Voltage Dependent ◽  
Cl Channels ◽  
Ca2 Channels

Background/Aims: Tetraethylammonium chloride (TEA) induces oscillatory contractions in mouse airway smooth muscle (ASM); however, the generation and maintenance of oscillatory contractions and their role in ASM are unclear. Methods: In this study, oscillations of ASM contraction and intracellular Ca2+ were measured using force measuring and Ca2+ imaging technique, respectively. TEA, nifedipine, niflumic acid, acetylcholine chloride, lithium chloride, KB-R7943, ouabain, 2-Aminoethoxydiphenyl borate, thapsigargin, tetrodotoxin, and ryanodine were used to assess the mechanism of oscillatory contractions. Results: TEA induced depolarization, resulting in activation of L-type voltage-dependent Ca2+ channels (LVDCCs) and voltage-dependent Na+ (VNa) channels. The former mediated Ca2+ influx to trigger a contraction and the latter mediated Na+ entry to enhance the contraction via activating LVDCCs. Meanwhile, increased Ca2+-activated Cl- channels, inducing depolarization that resulted in contraction through LVDCCs. In addition, the contraction was enhanced by intracellular Ca2+ release from Ca2+ stores mediated by inositol (1,4,5)-trisphosphate receptors (IP3Rs). These pathways together produce the contractile phase of the oscillatory contractions. Furthermore, the increased Ca2+ activated the Na+-Ca2+ exchanger (NCX), which transferred Ca2+ out of and Na+ into the cells. The former induced relaxation and the latter activated Na+/K+-ATPase that induced hypopolarization to inactivate LVDCCs causing further relaxation. This can also explain the relaxant phase of the oscillatory contractions. Moreover, the depolarization induced by VNa channels and NCX might be greater than the hypopolarization caused by Na+/K+-ATPase alone, inducing LVDCC activation and resulting in further contraction. Conclusions: These data indicate that the TEA-induced oscillatory contractions were cooperatively produced by LVDCCs, VNa channels, Ca2+-activated Cl- channels, NCX, Na+/K+ ATPase, IP3Rs-mediated Ca2+ release, and extracellular Ca2+.

scholarly journals Divergent Cl- and H+ pathways underlie transport coupling and gating in CLC exchangers and channels

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lilia Leisle ◽  
Yanyan Xu ◽  
Eva Fortea ◽  
Sangyun Lee ◽  
Jason D Galpin ◽  
...  
Keyword(s):  
Genetic Disorders ◽  
Md Simulations ◽  
Conformational Flexibility ◽  
Gating Mechanism ◽  
Ping Pong ◽  
Evolutionarily Conserved ◽  
Electrophysiological Measurements ◽  
Voltage Dependent ◽  
Cl Channels ◽  
Aromatic Pathway

The CLC family comprises H+-coupled exchangers and Cl- channels, and mutations causing their dysfunction lead to genetic disorders. The CLC exchangers, unlike canonical 'ping-pong' antiporters, simultaneously bind and translocate substrates through partially congruent pathways. How ions of opposite charge bypass each other while moving through a shared pathway remains unknown. Here, we use MD simulations, biochemical and electrophysiological measurements to identify two conserved phenylalanine residues that form an aromatic pathway whose dynamic rearrangements enable H+ movement outside the Cl- pore. These residues are important for H+ transport and voltage-dependent gating in the CLC exchangers. The aromatic pathway residues are evolutionarily conserved in CLC channels where their electrostatic properties and conformational flexibility determine gating. We propose that Cl- and H+ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters and suggest a unifying mechanism that describes the gating mechanism of both CLC subtypes.

scholarly journals Bimodal Control of a Ca2+-Activated Cl− Channel by Different Ca2+ Signals

1999 ◽  
Vol 115 (1) ◽  
pp. 59-80 ◽  
Author(s):  
Akinori Kuruma ◽  
H. Criss Hartzell
Keyword(s):  
Muscle Tone ◽  
Outward Current ◽  
Single Type ◽  
Channel Activation ◽  
Time Constants ◽  
Cl Channel ◽  
Physiological Processes ◽  
Steady State Current ◽  
Voltage Dependent ◽  
Cl Channels

Ca2+-activated Cl− channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca2+ and voltage. Excised inside-out patches containing many Ca2+-activated Cl− channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl− channel that exhibited an anionic selectivity of I− > Br− > Cl− (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca2+]. However, depending on the amplitude of the Ca2+ signal, this channel exhibited qualitatively different behaviors. At [Ca2+] < 1 μM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current–voltage relationship was strongly outwardly rectifying. At higher [Ca2+], the currents did not rectify and were time independent. This difference in behavior at different [Ca2+] was explained by an apparent voltage-dependent Ca2+ sensitivity of the channel. At +120 mV, the EC50 for channel activation by Ca2+ was approximately fourfold less than at −120 mV (0.9 vs. 4 μM). Thus, at [Ca2+] < 1 μM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca2+ binding. The voltage-dependent Ca2+ sensitivity was explained by a kinetic gating scheme in which channel activation was Ca2+ dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca2+ concentration jumps were voltage sensitive, but that the activation time constants were Ca2+ sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca2+ concentrations adds a new dimension to Ca2+ signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca2+ signal.

Intracellular ramification of endothelin signal

1991 ◽  
Vol 260 (5) ◽  
pp. C982-C992 ◽  
Author(s):  
K. Iijima ◽  
L. Lin ◽  
A. Nasjletti ◽  
M. S. Goligorsky
Keyword(s):  
Channel Blocker ◽  
Vascular Ring ◽  
Membrane Depolarization ◽  
Free Medium ◽  
Induced Membrane ◽  
Fura 2 ◽  
Inositol 1,4,5 Trisphosphate ◽  
Voltage Dependent ◽  
Cl Channels ◽  
Ca2 Channels

Effects of porcine 1-21 endothelin (ET-1) on [Ca2+]i, [Na+]i, and [Cl-]i and on membrane potential were studied in individual mesangial (MC) and vascular smooth muscle (VSMC) cells using microspectrofluorimetry of fura-2, SBFI, SPQ, and bis-oxonol, respectively. ET-1 increased [Ca2+]i by fivefold, showing an immediate and a sustained phase of response. Ca(2+)-free medium and nifedipine pretreatment significantly curtailed the sustained phase of response to ET-1. These findings were confirmed in studies of vascular ring preparations, demonstrating that Ca2+ influx may account for at least 50% of contraction. ET-1 caused immediate and sustained depolarization of MC and VSMC. This could not be attributed to Na+ influx, since fluorescence of SBFI was not affected by ET-1 and Na(+)-free medium did not abolish the ET-1-induced membrane depolarization. Studies of SPQ fluorescence changes induced by ET-1 revealed an increase in fluorescence intensity consistent with the decrease in [Cl-]i. A Cl- channel blocker, IAA-94, abolished changes in SPQ fluorescence and curtailed sustained phases of membrane depolarization and [Ca2+]i elevation in response to ET-1, but did not affect KCl-induced [Ca2+]i transients. IAA-94 also attenuated the ET-1-induced contraction of aortic rings. Microinjection of either calcium gluconate or inositol 1,4,5-trisphosphate (IP3) in SPQ-loaded cells resulted in an increase in fluorescence mimicking the effect of ET-1. These changes were blunted by pretreatment of cells with BAPTA and incubation in Ca(2+)-free medium. When IP3 was microinjected into fura-2-loaded MC, this resulted in immediate and sustained elevation of [Ca2+]i. In conclusion, generation of IP3 results in mobilization of intracellular Ca2+ stores and activation of Cl- channels. Ensuing Cl- efflux causes membrane depolarization and, in turn, activation of voltage-dependent Ca2+ channels, resulting in sustained elevation of [Ca2+]i which is indispensable for the full-scale contraction produced by ET-1.

Sign in / Sign up

Export Citation Format

Share Document