scholarly journals Voltage-dependent inactivation of T-tubular skeletal calcium channels in planar lipid bilayers.

1991 ◽  
Vol 97 (2) ◽  
pp. 393-412 ◽  
Author(s):  
R Mejía-Alvarez ◽  
M Fill ◽  
E Stefani
Keyword(s):  
Calcium Channels ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Channel Activity ◽  
T Tube ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Single Channel Activity ◽  
Kinetics Of ◽  
Channel Properties

Single-channel properties of dihydropyridine (DHP)-sensitive calcium channels isolated from transverse tubular (T-tube) membrane of skeletal muscle were explored. Single-channel activity was recorded in planar lipid bilayers after fusion of highly purified rabbit T-tube microsomes. Two populations of DHP-sensitive calcium channels were identified. One type of channel (noninactivating) was active (2 microM +/- Bay K 8644) at steady-state membrane potentials and has been studied in other laboratories. The second type of channel (inactivating) was transiently activated during voltage pulses and had a very low open probability (Po) at steady-state membrane potentials. Inactivating channel activity was observed in 47.3% of the experiments (n = 84 bilayers). The nonstationary kinetics of this channel was determined using a standard voltage pulse (HP = -50 mV, pulse to 0 mV). The time constant (tau) of channel activation was 23 ms. During the mV). The time constant (tau) of channel activation was 23 ms. During the pulse, channel activity decayed (inactivated) with a tau of 3.7 s. Noninactivating single-channel activity was well described by a model with two open and two closed states. Inactivating channel activity was described by the same model with the addition of an inactivated state as proposed for cardiac muscle. The single-channel properties were compared with the kinetics of DHP-sensitive inward calcium currents (ICa) measured at the cellular level. Our results support the hypothesis that voltage-dependent inactivation of single DHP-sensitive channels contributes to the decay of ICa.

scholarly journals Amino acid substitutions in the FXYD motif enhance phospholemman-induced modulation of cardiac L-type calcium channels

2010 ◽  
Vol 299 (5) ◽  
pp. C1203-C1211 ◽  
Author(s):  
Kai Guo ◽  
Xianming Wang ◽  
Guofeng Gao ◽  
Congxin Huang ◽  
Keith S. Elmslie ◽  
...  
Keyword(s):  
Amino Acid ◽  
Calcium Channels ◽  
Molecular Mechanisms ◽  
Channel Gating ◽  
Fine Tuning ◽  
Amino Acid Substitutions ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Kinetics Of

We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149–1159, 2010). The short 17 amino acid extracellular NH2-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate CaV1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with CaV1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on CaV1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca2+ dynamics in the heart.

scholarly journals A Gastropod Toxin Selectively Slows Early Transitions in the Shaker K Channel's Activation Pathway

2004 ◽  
Vol 123 (6) ◽  
pp. 685-696 ◽  
Author(s):  
Jon T. Sack ◽  
Richard W. Aldrich ◽  
William F. Gilly
Keyword(s):  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Gating Currents ◽  
K Channels ◽  
Voltage Sensors ◽  
Essentially Normal ◽  
Channel Opening ◽  
Single Channel Activity ◽  
Kinetics Of

A toxin from a marine gastropod's defensive mucus, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT), was found to inhibit voltage-gated potassium channels by a novel mechanism. Voltage-clamp experiments with Shaker K channels reveal that externally applied BrMT slows channel opening but not closing. BrMT slows K channel activation in a graded fashion: channels activate progressively slower as the concentration of BrMT is increased. Analysis of single-channel activity indicates that once a channel opens, the unitary conductance and bursting behavior are essentially normal in BrMT. Paralleling its effects against channel opening, BrMT greatly slows the kinetics of ON, but not OFF, gating currents. BrMT was found to slow early activation transitions but not the final opening transition of the Shaker ILT mutant, and can be used to pharmacologically distinguish early from late gating steps. This novel toxin thus inhibits activation of Shaker K channels by specifically slowing early movement of their voltage sensors, thereby hindering channel opening. A model of BrMT action is developed that suggests BrMT rapidly binds to and stabilizes resting channel conformations.

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.

scholarly journals The heterogeneity of ion channels in chromaffin granule membranes

2006 ◽  
Vol 11 (3) ◽  
Author(s):  
Renata Hordejuk ◽  
Adam Szewczyk ◽  
Krzysztof Dołowy
Keyword(s):  
Lipid Bilayers ◽  
Adrenal Glands ◽  
Single Channel ◽  
Ion Homeostasis ◽  
Planar Lipid Bilayers ◽  
Chromaffin Granules ◽  
Channel Activity ◽  
Chromaffin Granule ◽  
Catecholamine Synthesis ◽  
Single Channel Activity

AbstractChromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic 86Rb+ flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 ± 10 pS, 220 ± 8 pS, 152 ± 8 pS and 13 ± 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 ± 8 pS and 31 ± 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0.

Activation of cardiac ryanodine receptors by cardiac glycosides

2002 ◽  
Vol 282 (3) ◽  
pp. H1118-H1126 ◽  
Author(s):  
Toshio Sagawa ◽  
Kazuko Sagawa ◽  
James E. Kelly ◽  
Robert G. Tsushima ◽  
J. Andrew Wasserstrom
Keyword(s):  
Lipid Bilayers ◽  
Cardiac Glycosides ◽  
Single Channel ◽  
Lower Sensitivity ◽  
Maximal Effect ◽  
Channel Activity ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Channel Activation ◽  
Single Channel Activity ◽  
Ryr2 Channel

This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of ∼0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 μM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 μM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.

scholarly journals Single Channel Properties of Rat Acid–sensitive Ion Channel-1α, -2a, and -3 Expressed in Xenopus Oocytes

2002 ◽  
Vol 120 (4) ◽  
pp. 553-566 ◽  
Author(s):  
Ping Zhang ◽  
Cecilia M. Canessa
Keyword(s):  
Ion Channels ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Acid Sensing Ion Channels ◽  
Voltage Dependent ◽  
Subconductance States ◽  
Kinetics Of ◽  
Channel Properties

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1α, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na+ are similar for the three types of channels (23–18 pS) and are not voltage dependent. However, ASIC1α exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1α and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1α, ASIC2a, and ASIC3 are activated by external protons with apparent pH50 of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1α and ASIC3 (2.0 and 4.5 s−1, respectively) but slow and only partial in ASIC2a (0.045 s−1). The response to external Ca2+ also differs: μM concentrations of extracellular Ca2+ are necessary for proton gating of ASIC3 (EC50 = 0.28 μM), whereas ASIC1α and ASIC2a do not require Ca2+. In addition, Ca2+ inhibits ASIC1α (KD = 9.2 ± 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca2+ permeability of ASIC1α is very small.

Single-channel properties of high- and low-voltage-activated calcium channels in rat pituitary melanotropic cells

1994 ◽  
Vol 71 (3) ◽  
pp. 840-855 ◽  
Author(s):  
J. A. Keja ◽  
K. S. Kits
Keyword(s):  
Calcium Channels ◽  
Kinetic Analysis ◽  
Calcium Current ◽  
Time Course ◽  
Single Channel ◽  
Low Voltage ◽  
Channel Activity ◽  
Open Probability ◽  
Test Pulse ◽  
Channel Properties

1. Single-channel properties of voltage-dependent calcium channels were investigated in rat melanotropes in short-term primary culture. Unitary currents were resolved using the cell-attached configuration. 2. Depolarizations higher than -50 mV activated a population of 8.1-pS calcium channels [low-voltage activated (LVA)]. The LVA channel ensembles displayed a monoexponential time course of inactivation and a sigmoidal time course of activation fitted best by an m2h Hodgkin-Huxley-type model. Microscopic kinetic analysis suggested that at least one open state, two closed states, and one inactivated state are involved in channel gating. 3. At potentials positive to -20 mV a second class of calcium channels was activated with a conductance of 24.7 pS [high-voltage activated (HVA)]. HVA channels display different gating modes. Gating with high open probability (mode 2) and low open probability (mode 1) as well as blank traces (mode 0) are observed. The HVA channels were heterogeneous with respect to their inactivation properties. Ensembles that decayed entirely during a 300-ms test pulse as well as nondecaying ensembles were observed. Both HVA channel subtypes displayed sigmoidal activation, which was fitted by an m2 model. Microscopic kinetic analysis suggested that at least one open state and two closed states are involved in mode two gating of both HVA channel subtypes. 4. Depolarizing prepulses did not recruit or facilitate calcium channel activity in response to a test pulse, but inactivating HVA channel activity was strongly reduced. Depolarizing prepulses (+50 mV) did not affect the probability of opening of the noninactivating HVA channel. 5. The voltage dependence and kinetics of the LVA as well as both HVA channels are in good agreement with previously published data on the properties of the various calcium current components derived from whole-cell recordings of rat melanotropes. The data suggest that a T-type as well as two L-type channels (an inactivating and noninactivating channel) underlie the calcium current in these cells.

A calcium-activated chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

1996 ◽  
Vol 270 (6) ◽  
pp. C1675-C1686 ◽  
Author(s):  
J. I. Kourie ◽  
D. R. Laver ◽  
G. P. Ahern ◽  
A. F. Dulhunty
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Chloride Channel ◽  
Single Channel ◽  
Single Channels ◽  
Channel Activity ◽  
Open Probability ◽  
Single Channel Activity ◽  
Maximum Conductance

A Ca(2+)-activated Cl- channel is described in sarcoplasmic reticulum (SR) enriched vesicles of skeletal muscle incorporated into lipid bilayers. Small chloride (SCl) channels (n = 20) were rapidly and reversibly activated when cis- (cytoplasmic) [Ca2+] was increased above 10(-7) M, with trans-(luminal) [Ca2+] at either 10(-3) or 10(-7) M. The open probability of single channels increased from zero when cis-[Ca2+] was 10(-7) M to 0.61 +/- 0.12 when [Ca2+] was 10(-4) M. High- and low-conductance levels in single-channel activity were activated at different cis-[Ca2+]. Channel openings to the maximum conductance, 65-75 pS (250/50 mM Cl-, cis/ trans), were active when cis-[Ca2+] was increased above 5 x 10(-6) M. In contrast to the maximum conductance, channel openings to submaximal levels between 5 and 40 pS were activated at a lower cis-[Ca2+] and dominated channel activity between 5 x 10(-7) and 5 x 10(-6) M. Activation of SCl channels was Ca2+ specific and not reproduced by cytoplasmic Mg2+ concentrations of 10(-3) M. We suggest that the SCl channel arises in the SR membrane. The Ca2+ dependence of this channel implies that it is active at [Ca2+] achieved during muscle contraction.

scholarly journals Long-Term and Immediate Effect of Testosterone on Single T-Type Calcium Channel in Neonatal Rat Cardiomyocytes

Endocrinology ◽  
2006 ◽  
Vol 147 (11) ◽  
pp. 5160-5169 ◽  
Author(s):  
Guido Michels ◽  
Fikret Er ◽  
Michael Eicks ◽  
Stefan Herzig ◽  
Uta C. Hoppe
Keyword(s):  
Calcium Channels ◽  
Single Channel ◽  
Heart Diseases ◽  
Calcium Currents ◽  
Neonatal Rat ◽  
Channel Activity ◽  
Open Probability ◽  
Rat Cardiomyocytes ◽  
Single Channel Activity

In the cardiovascular system, T-type calcium channels play an important role for the intracellular calcium homeostasis and spontaneous pacemaker activity and are involved in the progression of structural heart diseases. Androgens influence the cardiovascular physiology and pathophysiology. However, their effect on native T-type calcium currents (ICa,T) remains unclear. To test the chronic effect of testosterone on the cardiac ICa,T, cultured neonatal rat ventricular cardiomyocytes were treated with testosterone (1 nm-10 μm) for 24–30 h. Current measurements were performed after testosterone washout to exclude any acute testosterone effects. Testosterone (100 nm) pretreatment significantly increased whole-cell ICa,T density from 1.26 ± 0.48 pA/pF (n = 8) to 5.06 ± 1.75 pA/pF (n = 7; P < 0.05) and accelerated beating rate. This was attributed to both increased expression levels of the pore-forming subunits Cav3.1 and Cav3.2 and increased T-type single-channel activity. On single-channel level, the increase of the ensemble average current by testosterone vs. time-matched controls was due to an increased availability (58.1 ± 4.2 vs. 21.5 ± 4.0%, P < 0.01) and open probability (2.78 ± 0.29 vs. 0.85 ± 0.23%, P < 0.01). Cotreatment with the selective testosterone receptor antagonist flutamide (10 μm) prevented these chronic testosterone-induced effects. Conversely, acute application of testosterone (10 μm) decreased T-type single-channel activity in testosterone pretreated cells by reducing the open probability (0.78 ± 0.13 vs. 2.91 ± 0.38%, P < 0.01), availability (23.6 ± 3.3 vs. 57.6 ± 4.5%, P < 0.01), and peak current (−20 ± 4 vs. −58 ± 4 fA, P < 0.01). Flutamide (10 μm) did not abolish the testosterone-induced acute block of T-type calcium channels. Our results indicate that long-term testosterone treatment increases, whereas acute testosterone decreases neonatal rat T-type calcium currents. These effects seem to be mediated by a genomic chronic stimulation and a nongenomic acute inhibitory action.

scholarly journals Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. Evidence for two voltage-dependent Ca2+ binding reactions.

1983 ◽  
Vol 82 (4) ◽  
pp. 511-542 ◽  
Author(s):  
E Moczydlowski ◽  
R Latorre
Keyword(s):  
Linear Function ◽  
Lipid Bilayers ◽  
Single Channel ◽  
K Channel ◽  
Planar Bilayers ◽  
Gating Kinetics ◽  
Rat Muscle ◽  
Voltage Dependent ◽  
The Mean ◽  
Kinetics Of

The gating kinetics of a Ca2+-activated K+ channel from adult rat muscle plasma membrane are studied in artificial planar bilayers. Analysis of single-channel fluctuations distinguishes two Ca2+- and voltage-dependent processes: (a) short-lived channel closure (less than 1 ms) events appearing in a bursting pattern; (b) opening and closing events ranging from one to several hundred milliseconds in duration. The latter process is studied independently of the first and is denoted as the primary gating mode. At constant voltage, the mean open time of the primary gating mode is a linear function of the [Ca2+], whereas the mean closed time is a linear function of the reciprocal [Ca2+]. In the limits of zero and infinite [Ca2+], the mean open and the mean closed times are, respectively, independent of voltage. These results are predicted by a kinetic scheme consisting of the following reaction steps: (a) binding of Ca2+ to a closed state; (b) channel opening; (c) binding of a second Ca2+ ion. In this scheme, the two Ca2+ binding reactions are voltage dependent, whereas the open-closed transition is voltage independent. The kinetic constant derived for this scheme gives an accurate theoretical fit to the observed equilibrium open-state probability. The results provide evidence for a novel regulatory mechanism for the activity of an ion channel: modulation by voltage of the binding of an agonist molecule, in this case, Ca2+ ion.

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