scholarly journals Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane.

1988 ◽  
Vol 92 (1) ◽  
pp. 67-86 ◽  
Author(s):  
A Oberhauser ◽  
O Alvarez ◽  
R Latorre
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Divalent Cations ◽  
Hill Coefficient ◽  
Muscle Membrane ◽  
K Channel ◽  
Single Channel Currents ◽  
The Hill ◽  
Channel Currents ◽  
Cytoplasmic Side

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.

Characterization of single non-inactivating potassium channels in primary neuronal cultures of Drosophila

1989 ◽  
Vol 145 (1) ◽  
pp. 173-184
Author(s):  
D. Yamamoto ◽  
N. Suzuki
Keyword(s):  
Time Distribution ◽  
Single Channel ◽  
K Channel ◽  
Delayed Rectifier ◽  
Neuronal Cultures ◽  
Patch Clamp Technique ◽  
Primary Neuronal Cultures ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Cytoplasmic Side

Permeability and gating properties of single, non-inactivating, K+ channel currents in cultured Drosophila neurons were studied using the gigaohm-seal patch-clamp technique. The non-inactivating K+ currents were activated by depolarizing the membrane to −30 mV or to more positive potentials. The slope conductance of the channel was estimated to be 17.6 +/− 3.70 pS when the cytoplasmic side of the inside-out membrane patch was perfused with solutions containing 145 mmoll-1 K+. The single-channel conductance was temperature-sensitive, with a Q10 of 1.44 between 10 and 20 degrees C. Single-channel currents could be recorded when the cytoplasmic K+ was replaced with NH4+, Rb+ or Na+, but not with Cs+. The conductance ratio of the channel for these cations was: K+ (1) greater than NH4+(0.53) greater than Rb+ (0.47) greater than Na+ (0.44). Tetraethylammonium (TEA+) ions applied at a concentration of 10 mmoll-1 to the cytoplasmic side of the membrane increased the frequency of ‘blank’ traces which contained no channel openings during repetitive depolarization. In addition, single-channel amplitude was reduced by about 20%. The open-time distribution was fitted by a single exponential function, whereas the closed-time distribution required a three-exponential fit. Permeability and gating properties of single, non-inactivating K+ channel currents in neurons of eag, a mutant which has defects in the delayed rectifier K+ channel, were indistinguishable from those recorded from wild-type neurons.

Intracellular ADP-ribose inhibits ATP-sensitive K+ channels in rat ventricular myocytes

1996 ◽  
Vol 271 (2) ◽  
pp. C464-C468 ◽  
Author(s):  
Y. G. Kwak ◽  
S. K. Park ◽  
U. H. Kim ◽  
M. K. Han ◽  
J. S. Eun ◽  
...  
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Physiological Role ◽  
Hill Coefficient ◽  
K Channel ◽  
Channel Activity ◽  
Rat Ventricular Myocytes ◽  
Cyclic Adp Ribose ◽  
The Hill

Cyclic ADP-ribose (cADPR), an NAD metabolite, has been shown to be a messenger for Ca2+ mobilization from intracellular Ca2+ stores. However, the physiological role of ADP-ribose (ADPR), another metabolite of NAD, is not known. We examined the effects of cADPR and ADPR on the ATP-sensitive K+ channel (KATP) activity in rat ventricular myocytes by use of the inside-out patch-clamp configuration. ADPR, but not cADPR, inhibited the channel activity at micromolar range with an inhibitor constant (Ki) of 38.4 microM. The Hill coefficient was 0.9. ATP inhibited the K+ channel with a Ki of 77.8 microM, and the Hill coefficient was 1.8. Single-channel conductance was not affected by ADPR. These findings strongly suggest that ADPR may act as a regulator of KATP channel activity.

scholarly journals Divalent Cation Interactions with Light-Dependent K Channels

1999 ◽  
Vol 114 (5) ◽  
pp. 653-672 ◽  
Author(s):  
Enrico Nasi ◽  
Maria del Pilar Gomez
Keyword(s):  
Binding Site ◽  
Single Channel ◽  
Divalent Cations ◽  
Light Response ◽  
Relaxation Methods ◽  
Physiological Conditions ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Single Binding Site

The light-dependent K conductance of hyperpolarizing Pecten photoreceptors exhibits a pronounced outward rectification that is eliminated by removal of extracellular divalent cations. The voltage-dependent block by Ca2+ and Mg2+ that underlies such nonlinearity was investigated. Both divalents reduce the photocurrent amplitude, the potency being significantly higher for Ca2+ than Mg2+ (K1/2 ≈ 16 and 61 mM, respectively, at Vm = −30 mV). Neither cation is measurably permeant. Manipulating the concentration of permeant K ions affects the blockade, suggesting that the mechanism entails occlusion of the permeation pathway. The voltage dependency of Ca2+ block is consistent with a single binding site located at an electrical distance of δ ≈ 0.6 from the outside. Resolution of light-dependent single-channel currents under physiological conditions indicates that blockade must be slow, which prompted the use of perturbation/relaxation methods to analyze its kinetics. Voltage steps during illumination produce a distinct relaxation in the photocurrent (τ = 5–20 ms) that disappears on removal of Ca2+ and Mg2+ and thus reflects enhancement or relief of blockade, depending on the polarity of the stimulus. The equilibration kinetics are significantly faster with Ca2+ than with Mg2+, suggesting that the process is dominated by the “on” rate, perhaps because of a step requiring dehydration of the blocking ion to access the binding site. Complementary strategies were adopted to investigate the interaction between blockade and channel gating: the photocurrent decay accelerates with hyperpolarization, but the effect requires extracellular divalents. Moreover, conditioning voltage steps terminated immediately before light stimulation failed to affect the photocurrent. These observations suggest that equilibration of block at different voltages requires an open pore. Inducing channels to close during a conditioning hyperpolarization resulted in a slight delay in the rising phase of a subsequent light response; this effect can be interpreted as closure of the channel with a divalent ion trapped inside.

scholarly journals Allosteric effects of Mg2+ on the gating of Ca2+-activated K+ channels from mammalian skeletal muscle

1986 ◽  
Vol 124 (1) ◽  
pp. 5-13
Author(s):  
J. Golowasch ◽  
A. Kirkwood ◽  
C. Miller
Keyword(s):  
Binding Sites ◽  
Hill Coefficient ◽  
K Channel ◽  
Activation Process ◽  
Mammalian Skeletal Muscle ◽  
Activation Curve ◽  
K Channels ◽  
The Hill ◽  
Cytoplasmic Side ◽  
High Conductance

Ca2+-activated K+ channels from rat muscle transverse tubule membranes were inserted into planar phospholipid bilayers, and the activation of these channels by Ca2+ was studied. On the cytoplasmic side of the channel, calcium ions (in the range 10–100 mumol l-1) increase the opening probability of the channel in a graded way. This ‘activation curve’ is sigmoid, with an average Hill coefficient of about 2. Magnesium ions, in the range 1–10 mmol l-1, increase the apparent affinity of the channel for Ca2+ and greatly enhance the sigmoidicity of the Ca2+ activation curve. In the presence of 10 mmol l-1 Mg2+, the Hill coefficient for Ca2+ activation is about 4.5. This effect depends upon Mg2+ concentration but not upon applied voltage. Mg2+ is effective only when added to the cytoplasmic side of the channel. The results argue that this high-conductance, Ca2+-activated K+ channel contains at least six Ca2+-binding sites involved in the activation process.

Anion channels for amino acids in MDCK cells

1992 ◽  
Vol 263 (6) ◽  
pp. C1200-C1207 ◽  
Author(s):  
U. Banderali ◽  
G. Roy
Keyword(s):  
Amino Acids ◽  
Single Channel ◽  
Mdck Cells ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Excised Patches ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Inside Out ◽  
Cytoplasmic Side

Large losses of amino acids by diffusion were previously observed in Madin-Darby canine kidney (MDCK) cells during volume regulation. Also, an outward rectifying anion channel was activated. Because this channel was not selective among anions, it was suggested that it could be permeable to amino acids. Its permeability to aspartate, glutamate, and taurine was studied using the patch-clamp technique in the inside-out configuration. Solutions containing 500 mM aspartate or glutamate were used on the cytoplasmic side of excised patches to detect single-channel currents carried by these anions. Permeability ratios were estimated in two different ways: 1) from the shift in reversal potential of current-voltage curves after anion replacement in the bath solution and 2) from comparisons of amplitudes of single-channel currents carried by tested anions and chloride, respectively. The values of aspartate-to-chloride and glutamate-to-chloride permeability ratios obtained with both methods were quite consistent and were of the order of 0.2 for both amino acids. Taurine in solutions at physiological pH 7.3 is a zwitterionic molecule and bears no net charge. To detect single-channel currents carried by taurine, solutions containing 500 mM taurine at pH 8.2 were used in inside-out experiments. Under these conditions 120 mM of negatively charged taurine was present in the solutions bathing the cytoplasmic side of excised patches. The permeability ratio estimated from the shift in reversal potential was 0.75. These results showed that some of the organic compounds released by cells during regulatory volume decrease could diffuse through this outwardly rectifying anionic channel.

scholarly journals Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers.

1987 ◽  
Vol 90 (3) ◽  
pp. 375-395 ◽  
Author(s):  
E Recio-Pinto ◽  
D S Duch ◽  
S R Levinson ◽  
B W Urban
Keyword(s):  
Sodium Channel ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Planar Bilayers ◽  
Electric Eel ◽  
Voltage Dependent ◽  
Single Channel Activity ◽  
Single Channel Currents ◽  
Channel Currents

Highly purified sodium channel protein from the electric eel, Electrophorus electricus, was reconstituted into liposomes and incorporated into planar bilayers made from neutral phospholipids dissolved in decane. The purest sodium channel preparations consisted of only the large, 260-kD tetrodotoxin (TTX)-binding polypeptide. For all preparations, batrachotoxin (BTX) induced long-lived single-channel currents (25 pS at 500 mM NaCl) that showed voltage-dependent activation and were blocked by TTX. This block was also voltage dependent, with negative potentials increasing block. The permeability ratios were 4.7 for Na+:K+ and 1.6 for Na+:Li+. The midpoint for steady state activation occurred around -70 mV and did not shift significantly when the NaCl concentration was increased from 50 to 1,000 mM. Veratridine-induced single-channel currents were about half the size of those activated by BTX. Unpurified, nonsolubilized sodium channels from E. electricus membrane fragments were also incorporated into planar bilayers. There were no detectable differences in the characteristics of unpurified and purified sodium channels, although membrane stability was considerably higher when purified material was used. Thus, in the eel, the large, 260-kD polypeptide alone is sufficient to demonstrate single-channel activity like that observed for mammalian sodium channel preparations in which smaller subunits have been found.

Single-channel and whole-cell currents evoked by acetylcholine in dissociated sympathetic neurons of the rat

1987 ◽  
Vol 232 (1267) ◽  
pp. 239-248 ◽  
Keyword(s):  
Single Channel ◽  
Divalent Cations ◽  
Sympathetic Neurons ◽  
Outward Current ◽  
Inward Rectification ◽  
Normal Medium ◽  
Whole Cell ◽  
Old Rats ◽  
Single Channel Currents ◽  
Channel Currents

Single acetylcholine-activated channels have been recorded from neurons dissociated from the sympathetic chain of 17–21 day old rats. The mean single channel conductance is 35 pS in normal medium containing 1 mM calcium, and 51 pS in the absence of calcium. The measured current amplitudes are about five times more variable than at the frog endplate, at least in part because the current, while the channel is open, is much noisier than when it is shut. Single activations of the receptor by acetylcholine (ACh) produce a burst of openings; the distribution of the burst length has two components, the longer of which is of primary importance in synaptic transmission. Whole-cell currents, in response to ACh (up to 30 μM), show strong inward rectification with no outward current being detectable. This phenomenon is similar whether the intracellular ion is sodium or cesium, whether or not divalent cations are present, and whether or not atropine is present. Nevertheless, outward single-channel currents (of normal conductance) are detectable in isolated outside-out patches.

scholarly journals Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel

2010 ◽  
Vol 135 (5) ◽  
pp. 509-526 ◽  
Author(s):  
Victor P.T. Pau ◽  
Karin Abarca-Heidemann ◽  
Brad S. Rothberg
Keyword(s):  
Lipid Bilayers ◽  
Complete Solution ◽  
Hill Coefficient ◽  
K Channel ◽  
Open Probability ◽  
Allosteric Coupling ◽  
Allosteric Mechanism ◽  
Channel Opening ◽  
Tenfold Increase ◽  
Cytoplasmic Side

MthK is a Ca2+-gated K+ channel whose activity is inhibited by cytoplasmic H+. To determine possible mechanisms underlying the channel’s proton sensitivity and the relation between H+ inhibition and Ca2+-dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca2+] (ranging from nominally 0 to 30 mM) at a given [H+], in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between [Ca2+] and open probability (Po), with a mean Hill coefficient (nH) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor nH changed significantly as a function of [H+] over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca2+ was not H+ sensitive over pH ranging from 7.3 to 9.0. However, increasing [H+] raised the EC50 for Ca2+ activation by ∼4.7-fold per tenfold increase in [H+], displaying a linear relation between log(EC50) and log([H+]) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H+ binding does not directly modulate either the channel’s closed–open equilibrium or the allosteric coupling between Ca2+ binding and channel opening. We can account for the Ca2+ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca2+ allosterically activates MthK, whereas H+ opposes activation by destabilizing the binding of Ca2+.

Oxantel-activated single channel currents in the muscle membrane of Ascaris suum

Parasitology ◽  
1995 ◽  
Vol 110 (4) ◽  
pp. 437-448 ◽  
Author(s):  
V. M. E. Dale ◽  
R. J. Martin
Keyword(s):  
Single Channel ◽  
Ascaris Suum ◽  
Muscle Membrane ◽  
Kinetic Properties ◽  
Voltage Sensitivity ◽  
Channel Block ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Single Channel Currents ◽  
Channel Currents

SUMMARYThe patch clamp technique was used to investigate the action of the anthelmintic drug, oxantel, on nicotinic acetylcholine receptor (nAChR) currents recorded from vesicles of the somatic muscle cells of the nematode parasite Ascaris suum. The amplitudes of the currents were analysed at different membrane potentials to determine the single channel conductance. Also the open and closed durations were measured to determine the kinetic properties of the activated channel. Oxantel activated single nAChR currents throughout a concentration range 10–100 μM, these currents were not observed with oxantel-free pipette solutions. The mean open time of the activated channels at a membrane potential of –75 mV and a concentration of 10 μM was 1·34 ms. At higher concentrations the open times were shorter and voltage sensitive, decreasing in duration on hyperpolarization, thus suggesting open channel block. The kinetics were analysed using a simple channel block model. The forward block rate, K + B, increased with increasing oxantel concentration but showed little increase as the membrane was hyperpolarized. K + B was 2·41×107 M−1s−1 – 50 mV and 2·64 × 107 M−1s−1 at – 100mV. The unblocking rate constant, K – B, did exhibit voltage sensitivity being 443·6 s−1 at – 50 mV and 86·8 s−1 at –100 mV. Thus the blocking dissociation constant KB (= K – B/K + B) was 18·5 μM at –50 mV and 3·3 μM at –100 mV. The simple channel block scheme was found to be insufficient to explain fully the observations made; reasons for this are discussed.

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