scholarly journals Antibodies to the ciliary membrane of Paramecium tetraurelia alter membrane excitability.

1983 ◽  
Vol 97 (5) ◽  
pp. 1421-1428 ◽  
Author(s):  
R Ramanathan ◽  
Y Saimi ◽  
J B Peterson ◽  
D L Nelson ◽  
C Kung
Keyword(s):  
Antibody Binding ◽  
Voltage Sensitivity ◽  
Ca Channel ◽  
Paramecium Tetraurelia ◽  
Membrane Excitability ◽  
Ca Current ◽  
Antibody Treatment ◽  
Ciliary Membrane ◽  
Entire Cell ◽  
Electrophysiological Effects

Immobilization of Paramecium followed the binding of antibodies to the major proteins of the ciliary membrane (the immobilization antigens, i-antigens, approximately 250,000 mol wt). Immunoelectron microscopy showed this binding to be serotype-specific and to occur over the entire cell surface. Antibody binding also reduced the current through the Ca-channel of the excitable ciliary membrane as monitored using a voltage-clamp. The residual Ca-current appeared normal in its voltage sensitivity and kinetics. As a secondary consequence of antibody binding, the Ca-induced K-current was also reduced. The resting membrane characteristics and other activatable currents, however, were not significantly altered by the antibody treatment. Since monovalent fragments of the antibodies also reduced the current but did not immobilize the cell, the electrophysiological effects were not the secondary consequences of immobilization. Antibodies against the second most abundant family of proteins (42,000-45,000 mol wt) had similar electrophysiological effects as revealed by experiments in which the Paramecia and the serum were heterologous with respect to the i-antigen but homologous with respect to the 42,000-45,000-mol-wt proteins. Protease treatment, shown to remove the surface antigen, also caused a reduction of the Ca-inward current. The loss of the inward Ca-current does not seem to be due to a drop in the driving force for Ca++ entry since increasing the external Ca++ or reducing the internal Ca++ (through EGTA injection) did not restore the current. Here we discuss the possibilities that (a) the major proteins define the functional environment of the Ca-channel and that (b) the Ca-channel is more susceptible to certain general changes in the membrane.

Changes in intracellular pH affect calcium currents in Paramecium caudatum

1982 ◽  
Vol 216 (1203) ◽  
pp. 209-224 ◽  
Keyword(s):  
Membrane Potential ◽  
Intracellular Ph ◽  
Outward Current ◽  
Calcium Currents ◽  
Resting Membrane Potential ◽  
Ca Channel ◽  
Paramecium Caudatum ◽  
Membrane Excitability ◽  
Ca Current ◽  
Intracellular Alkalinization

The relation between intracellular pH and membrane excitability was studied in the holotrich ciliate Paramecium caudatum . Intracellular pH (pH j ) was measured with recessed-tip ion-sensitive microelectrodes (Thomas 1974) and electrical properties were examined by current stimulation and conventional two-electrode voltage clamp. Under normal conditions the resting pH i of Paramecium was 6.80 ± 0.05. Intracellular alkalinization enhanced the early Ca current, while internal acidification depressed the Ca current. Both effects occurred in a voltage-independent manner. The late outward current was relatively unaffected by these alterations. Results obtained with replacement of extracellular Ca 2+ by Ba 2+ also support a direct effect of pH i on current through the Ca channel. Intracellular alkalinization to pH 7.15 converted graded, quasi-regenerative Ca responses elicited by injected current pulses into all-or-none action potentials. This change to all-or-none behaviour is presumed to be due to the increase in Ca current and a consequent change in the balance of inward and outward currents. Extracellular pH changes had little effect on pH i , resting membrane potential or the current-voltage relations. The intracellular pH was also independent of shifts in membrane potential. The results are consistent with a model in which Ca channel permeability is blocked by intracellular protonation of a single titratable site having an apparent dissociation constant of 6.2.

scholarly journals An anticalmodulin drug, W-7, inhibits the voltage-dependent calcium current in Paramecium caudatum

1984 ◽  
Vol 110 (1) ◽  
pp. 169-181 ◽  
Author(s):  
T. M. Hennessey ◽  
C. Kung
Keyword(s):  
Voltage Sensitivity ◽  
Channel Blockers ◽  
Ca Channel ◽  
Dependent Manner ◽  
Ca Current ◽  
Concentration Dependent Manner ◽  
Ca Channel Blockers ◽  
Voltage Dependent ◽  
Behavioural Consequence ◽  
Effective Analysis

The anticalmodulin drug, W-7 [N-(6 aminohexyl)-5-chloro-1-naphthalenesulphonamide] specifically inhibits the voltage-dependent Ca-current of Paramecium as well as the behavioural consequence of Ca2+ influx, backward swimming. The dechlorinated analogue, W-5, is four to five times less effective. Analysis of membrane currents under voltage clamp shows that W-7 not only reversibly inhibits the voltage-dependent Ca-current but also shifts the voltage sensitivity of this Ca-current towards less negative voltages in a concentration-dependent manner. We suggest that Paramecium can be used as a system to screen behaviourally for other Ca-channel blockers as well as to study the mechanism of action of these drugs.

Sodium-calcium exchange-mediated contractions in feline ventricular myocytes

1992 ◽  
Vol 263 (4) ◽  
pp. H1161-H1169 ◽  
Author(s):  
H. B. Nuss ◽  
S. R. Houser
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Membrane Potentials ◽  
Exchange Mechanism ◽  
Ca Channel ◽  
Ca Current ◽  
Voltage Step ◽  
Reverse Mode ◽  
Sodium Calcium ◽  
Calcium Exchange

The hypothesis that Ca entry by the sarcolemmal Na-Ca exchange mechanism induces sarcoplasmic reticulum (SR) Ca release, loads the SR with Ca, and/or directly induces contractions by elevating cytosolic free Ca was tested in voltage-clamped feline ventricular myocytes. Intracellular Na concentration was increased by cellular dialysis to enhance Ca influx via "reverse-mode" Na-Ca exchange at positive membrane potentials, at which the "L-type" Ca current (ICa) should be small. Contractions were induced in the presence of Ca channel antagonists by depolarization to these potentials, suggesting that Ca influx via reverse-mode Na-Ca exchange was involved. These contractions had both phasic (SR related) and tonic components of shortening. They were smaller and began with more delay after depolarization than contractions which involved ICa. The magnitude of shortening was graded by the amount and duration of depolarization, suggesting that Ca influx via reverse-mode Na-Ca exchange has the capacity to induce and grade SR Ca release. Small slow contractions could be evoked in the presence of ryanodine (to impair SR function) and verapamil (to block ICa), supporting the idea that Ca influx via Na-Ca exchange is sufficient to directly activate the contractile proteins. Contractions induced by voltage steps to +10 mV, which were usually small when ICa was blocked, were potentiated if preceded by a voltage step to strongly positive potentials. This potentiation was inhibited by ryanodine, suggesting that Ca entry that occurs by Na-Ca exchange may be important for normal SR Ca loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage Sensitive Ca-Channels and the Transient Inward Current in Paramecium Tetraurelia

1979 ◽  
Vol 78 (1) ◽  
pp. 149-161 ◽  
Author(s):  
YOUKO SATOW ◽  
CHING KUNG
Keyword(s):  
Voltage Clamp ◽  
Resting Potential ◽  
Ca Channel ◽  
Paramecium Tetraurelia ◽  
Ca Channels ◽  
Action Current ◽  
Inward Current ◽  
Transient Inward Current ◽  
Inorganic Cation ◽  
Inward Currents

Transient inward currents across the membrane of P. tetraurelia are recorded upon step depolarizations with a voltage clamp in solutions where Ca2+ is the only added inorganic cation. It is shown that the current is normally carried by Ca2+ through the Ca-channels which activate and inactivate in time. The transient inward current is dependent on both the size of the depolarizing step and the holding level before the step. Maximum inward current (Imax) occurs when the membrane is first held at the resting level (- 30 mV), then stepped to 0 mV in a solution containing 0.91 mM-Ca2+. The Imax is smaller when the membrane is first held at depolarized level. This is due to the depolarization-sensitive inactivation of the Ca-channels. The Imax is also smaller when the membrane is first held at a hyperpolarized level. This may be explained by the activation of hyperpolarization-sensitive K-channels known to exist in the Paramecium membrane. I max increases with concentration of Ca2+ up to 0.9 mM. Further increase in the Ca2+ concentration does not affect Imax. This apparent saturation at 0.9 mM-Ca2+ may reflect a rate-limiting step of Ca2+ permeation. The increase in Ca2+ concentration shifts the V-Ipeak curve in the direction of less sensitivity. This result is best explained as the effect of bound Ca2+ on the surface potential of the Paramecium membrane. These results provide the first detailed description of the properties of the action current through the Ca-channel in Paramecium. They also define the conditions under which future voltage-clamp studies of wild-type and mutant membranes of P. tetraurelia should be performed, i.e. to maximize the resolution of the Ca-channel activity, the membrane should be held at or near the resting potential and there should be over 0.9 mM-Ca2+ in the test solutions. The behaviour of the Paramecium Ca-channel and small Imax in the presence of K+ are discussed.

scholarly journals Properties of L-type calcium channel gating current in isolated guinea pig ventricular myocytes.

1991 ◽  
Vol 98 (2) ◽  
pp. 265-285 ◽  
Author(s):  
R W Hadley ◽  
W J Lederer
Keyword(s):  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Channel Gating ◽  
Charge Movement ◽  
Ca Channel ◽  
Dependent Manner ◽  
Gating Current ◽  
Ca Current ◽  
Channel Inactivation ◽  
Time Dependencies

Nonlinear capacitative current (charge movement) was compared to the Ca current (ICa) in single guinea pig ventricular myocytes. It was concluded that the charge movement seen with depolarizing test steps from -50 mV is dominated by L-type Ca channel gating current, because of the following observations. (a) Ca channel inactivation and the immobilization of the gating current had similar voltage and time dependencies. The degree of channel inactivation was directly proportional to the amount of charge immobilization, unlike what has been reported for Na channels. (b) The degree of Ca channel activation was closely correlated with the amount of charge moved at all test potentials between -40 and +60 mV. (c) D600 was found to reduce the gating current in a voltage- and use-dependent manner. D600 was also found to induce "extra" charge movement at negative potentials. (d) Nitrendipine reduced the gating current in a voltage-dependent manner (KD = 200 nM at -40 mV). However, nitrendipine did not increase charge movement at negative test potentials. Although contamination of the Ca channel gating current from other sources cannot be fully excluded, it was not evident in the data and would appear to be small. However, it was noted that the amount of Ca channel gating charge was quite large compared with the magnitude of the Ca current. Indeed, the gating current was found to be a significant contaminant (19 +/- 7%) of the Ca tail currents in these cells. In addition, it was found that Ca channel rundown did not diminish the gating current. These results suggest that Ca channels can be "inactivated" by means that do not affect the voltage sensor.

Voltage-dependent calcium current and the effects of adrenergic modulation in rat aortic smooth muscle cells

1992 ◽  
Vol 337 (1279) ◽  
pp. 37-47 ◽  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Ca Channel ◽  
Ca Current ◽  
Inward Current ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Inward Currents ◽  
Sustained Inward Current

Smooth muscle cells from rat aorta were cultured in defined, serum-free medium and studied using whole-cell patch-clamp techniques. Under conditions designed to isolate currents through Ca channels, step depolarizations produced inward currents which were fast in onset and inactivated rapidly, with little sustained inward current being observed. Both Ni and Cd blocked these currents, with Ni being effective at 50 μM. Removal of external Na or addition of 1 μM tetrodotoxin had no effect. Peak inward currents were attained at about —15 mV, with half-maximal activation at —41 mV using —80 mV holding potentials. The transient inward currents were reduced by depolarized holding potentials, with half-maximal steady-state inactivation at —48 mV. In three of the 98 cells studied, small maintained inward currents were observed with a —40 mV holding potential. The Ca channel antagonist nicardipine (5 μM ) blocked the transient inward current while neither of the dihydropyridine Ca channel agonists S( + )202 791 and ( — )BAY K 8644 produced a significant augmentation of sustained inward current. At 10 μM, both noradrenaline and adrenaline but not phenylephrine decreased the peak inward current. This inhibition was unaffected by a variety of adrenoceptor antagonists and was also observed when internal solutions having high Ca buffering capacity were used, but was absent when GDP-β-S instead of GTP was included in the pipette solution. The main conclusions from this study are that under our cell culture conditions, rat aortic smooth muscle cells possess predominately a transient, low-threshold-activated inward Ca current and that this Ca current is inhibited by certain adrenoceptor agonists but with a quite atypical adrenoceptor antagonist pharmacology.

A mathematical model of phase 2 reentry: role of L-type Ca current

2003 ◽  
Vol 284 (4) ◽  
pp. H1285-H1294 ◽  
Author(s):  
Shunichiro Miyoshi ◽  
Hideo Mitamura ◽  
Kana Fujikura ◽  
Yukiko Fukuda ◽  
Kojiro Tanimoto ◽  
...  
Keyword(s):  
Mathematical Model ◽  
Brugada Syndrome ◽  
Field Potential ◽  
Outward Current ◽  
Transient Outward Current ◽  
Ca Channel ◽  
Phase 2 ◽  
Ca Current ◽  
St Segment Elevation ◽  
St Segment

Phase 2 reentry (P2R) is known to be one of the mechanisms of malignant ventricular arrhythmias, especially those associated with Brugada syndrome. However, little is known about the underlying mechanism for P2R. Our aim in this study was to simulate P2R in a mathematical model to enable us to understand its mechanism and identify a potential therapeutic target. A mathematical model of the L-type Ca current was composed according to whole cell current data from guinea pig ventricular myocytes recorded at 37°C. Our mathematical model was incorporated into the modified Luo-Rudy phase 2 model. We set a dispersion in transient outward current ( I to) density within the theoretical fiber, composed of 80 serially arranged epicardial cells with gap junctions and then observed the P2R. The dispersion in I todensity within an only 0.8-cm epicardial theoretical fiber generated P2R with our Ca channel but not with the original model. When the P2R developed in the theoretical fiber, the calculated extracellular field potential showed coved-type ST segment elevation. We succeeded in generating P2R in our model for the first time. The local epicardial P2R may contribute the genesis of coved-type ST segment elevation in the Brugada syndrome.

scholarly journals Ionic regulation of adenylate cyclase from the cilia of Paramecium tetraurelia

1987 ◽  
Vol 246 (1) ◽  
pp. 187-192 ◽  
Author(s):  
J E Schultz ◽  
D G Uhl ◽  
S Klumpp
Keyword(s):  
Adenylate Cyclase ◽  
Kinetic Analysis ◽  
Binding Site ◽  
Dissociation Constant ◽  
Metal Binding ◽  
Paramecium Tetraurelia ◽  
Ionic Regulation ◽  
Metal Binding Site ◽  
Ciliary Membrane ◽  
Kinetics Of

The kinetics of the ionic regulation of an adenylate cyclase associated with the excitable ciliary membrane from Paramecium tetraurelia was examined. Glycerol (30%, v/v) stabilized the enzyme, and activated by an increase in Vmax. (3-fold) and a decrease in the apparent Km for MgATP (6-fold). Kinetic analysis of Mg2+ effects showed a stimulation via a single metal-binding site separate from the substrate site, with a dissociation constant, Ks, of 0.27 mM. Analysis of Ca2+ effects showed (i) an uncompetitive inhibition with respect to substrate MgATP, and (ii) dependence of the extent of inhibition on the free Mg2+ concentration. Ki values ranged from 4 to 130 microM-Ca2+ in the presence of 0.55-2 mM-Mg2+ respectively. This indicates competition between Mg2+ and Ca2+ at the metal-binding site. The Ca2+ effect was specific; Sr2+ and Ba2+ were almost without effect, and 100 microM-Ba2+ did not interfere with the Ca2+ inhibition. The actions of Ca2+ were readily reversible after addition of EGTA. K+ activated the adenylate cyclase at concentrations around 20 mM. The stimulatory potency of K+ was dependent on the free Mg2+ concentration. At 1 mM free Mg2+, 20 mM-K+ doubled the adenylate cyclase activity. The inhibitory Ca2+ and stimulatory K+ inputs were independent of each other.

A binding-site model for calcium channel inactivation that depends on calcium entry

1982 ◽  
Vol 217 (1206) ◽  
pp. 101-110 ◽  
Keyword(s):  
Membrane Surface ◽  
Calcium Entry ◽  
Ca Channel ◽  
Ca Channels ◽  
Ca Current ◽  
Two Phase ◽  
Site Model ◽  
Channel Inactivation ◽  
Calcium Channel Inactivation ◽  
Simplifying Assumptions

We describe a model for the mechanism by which Ca channel inactivation might depend on calcium entry. Ca is assumed to bind to a site at the internal membrane surface to cause inactivation of Ca channels. We assume that Ca that enters through the membrane accumulates in a submembrane compartment and also make simplifying assumptions about Ca buffering and removal. Our model predicts the results of single and double-pulse voltage-clamp experiments well. The predicted turn-off of Ca current is non-exponential. The model also predicts that procedures that slow inactivation will increase peak Ca current and suggests that both two-phase turn-off of currents and failure of normalized current to recover to 1. 0 in two-pulse experiments may be explained without assuming a voltage-dependent component of inactivation or two popu­lations of Ca channels.

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