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.

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.

Ca-dependent facilitation of cardiac Ca current is due to Ca-calmodulin-dependent protein kinase

1994 ◽  
Vol 267 (3) ◽  
pp. H982-H993 ◽  
Author(s):  
W. Yuan ◽  
D. M. Bers
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Ventricular Myocytes ◽  
Membrane Surface ◽  
Ca Channel ◽  
Ca Current ◽  
Dependent Protein Kinase ◽  
Inner Membrane ◽  
Recovery From Inactivation ◽  
Dependent Protein

Repetitive membrane potential (Em) depolarization from -90 to 0 mV in rabbit and ferret ventricular myocytes induces a facilitation or "staircase" of Ca current (ICa), which is Ca (not Em) dependent and takes several seconds to accumulate and dissipate. That is, ICa at the tenth pulse at 1-2 Hz exceeds that at the first pulse (I10 > I1). The ICa staircase was completely abolished by dialysis with either of two inhibitory peptides of Ca-calmodulin-dependent protein kinase (CaMKII) CaMKII(290-309) and CaMKII(273-302)], implicating this kinase. Inclusion of ATP gamma S in the patch pipette gradually increased ICa but also abolished the staircase implicating phosphorylation. KN-62, a nonpeptide CaMKII inhibitor, reversed the ICa staircase (I1 > I10). However, this effect of KN-62 was largely attributed to a slower recovery from inactivation and a gating shift to more negative Em (not seen with CaMKII peptides). Similar results were obtained with H-89 and staurosporine (inhibitors of adenosine 3',5'-cyclic monophosphate and phospholipid-/Ca-dependent protein kinase, respectively). The reversal of the ICa staircase with H-89 and KN-62 could be prevented by more negative interpulse Em or elevation of extracellular [Ca] (which could counteract changes in channel gating due to a reduction in internal negative surface potential). That is, these kinase inhibitors might decrease phosphorylation at the inner membrane surface. In approximately 30% of the cells studied with H-89 and staurosporine the characteristic kinetic difference in ICa inactivation (faster at I1 than I10) was also diminished. This might be due to a relatively nonspecific inhibition of the same protein kinase inhibited by the CaMKII peptides. We conclude that the Ca-dependent ICa facilitation is due to activation of CaMKII and phosphorylation of a site on or near the Ca channel. KN-62, H-89, and staurosporine shifted ICa gating to more negative potentials and slowed recovery from inactivation, effects that could be due to reduction in phosphorylation at the inner membrane surface. Thus the reversal of the ICa staircase by KN-62, H-89, and staurosporine may not be Ca channel specific.

scholarly journals Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium.

1994 ◽  
Vol 104 (5) ◽  
pp. 821-856 ◽  
Author(s):  
I Bezprozvanny ◽  
B E Ehrlich
Keyword(s):  
Ryanodine Receptor ◽  
Single Channel ◽  
Divalent Cations ◽  
Reversal Potential ◽  
Ca Channel ◽  
Ca Channels ◽  
Ca Current ◽  
Open Probability ◽  
Inositol 1,4,5 Trisphosphate ◽  
Conduction Properties

The conduction properties of inositol (1,4,5)-trisphosphate (InsP3)-gated calcium (Ca) channels (InsP3R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single-channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP3R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (approximately 5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single-ion pores. Measurements of InsP3R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (> or = 10 mM) decreased channel open probability 5-10-fold. When InsP3R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a PBa/PK of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP3R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP3R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltage-gated Ca channels of the plasma membrane. (b) The estimated size of the Ca current through the InsP3R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP3R by intraluminal Ca in the submillimolar range remains controversial. (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP3R activity was developed and the kinetic parameters of InsP3R gating were determined.

scholarly journals Two kinds of calcium channels in canine atrial cells. Differences in kinetics, selectivity, and pharmacology.

1985 ◽  
Vol 86 (1) ◽  
pp. 1-30 ◽  
Author(s):  
B P Bean
Keyword(s):  
Single Channel ◽  
Cardiac Cells ◽  
Fluctuation Analysis ◽  
Ca Channel ◽  
Ca Channels ◽  
Ca Current ◽  
Channel Current ◽  
Atrial Cells ◽  
Cell Recording ◽  
Ca Channel Current

Currents through Ca channels were recorded in single canine atrial cells using whole-cell recording with patch pipettes. Two components of Ca channel current could be distinguished. One ("Ifast") was present only if cells were held at negative potentials, was most prominent for relatively small depolarizations, and inactivated within tens of milliseconds. The other ("Islow"), corresponding to the Ca current previously reported in single cardiac cells, persisted even at relatively positive holding potentials, required stronger depolarizations for maximal current, and inactivated much more slowly. Both currents were unaffected by tetrodotoxin and both were reduced by Co. Ifast had the same size and kinetics when Ca was exchanged for Ba, while Islow was bigger and slower with Ba as the charge carrier. In isotonic BaCl2, fluctuation analysis showed that Ifast had a smaller single channel current than Islow. Islow was much more sensitive to block by nitrendipine than was Ifast; also, Islow, but not Ifast, was increased by the dihydropyridine drug BAY K8644. Isoproterenol produced large increases in Islow but had no effect on Ifast.

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)

scholarly journals Internal and external effects of dihydropyridines in the calcium channel of skeletal muscle.

1990 ◽  
Vol 95 (1) ◽  
pp. 1-27 ◽  
Author(s):  
H H Valdivia ◽  
R Coronado
Keyword(s):  
Skeletal Muscle ◽  
Lipid Bilayers ◽  
Radioligand Binding ◽  
Quantitative Agreement ◽  
Bay K 8644 ◽  
Single Channels ◽  
Ca Channel ◽  
Ca Channels ◽  
Receptor Sites ◽  
Internal Side

The agonist effect of the dihydropyridine (DHP) (-)Bay K 8644 and the inhibitory effects of nine antagonist DHPs were studied at a constant membrane potential of 0 mV in Ca channels of skeletal muscle transverse tubules incorporated into planar lipid bilayers. Four phenylalkylamines (verapamil, D600, D575, and D890) and d-cis-diltiazem were also tested. In Ca channels activated by 1 microM Bay K 8644, the antagonists nifedipine, nitrendipine, PN200-110, nimodipine, and pure enantiomer antagonists (+)nimodipine, (-)nimodipine, (+)Bay K 8644, inhibited activity in the concentration range of 10 nM to 10 microM. Effective doses (ED50) were 2 to 10 times higher when HDPs were added to the internal side than when added to the external side. This sidedness arises from different structure-activity relationships for DHPs on both sides of the Ca channel since the ranking potency of DHPs is PN200-110 greater than (-)nimodipine greater than nifedipine approximately S207-180 on the external side while PN200-110 greater than S207-180 greater than nifedipine approximately (-)nimodipine on the internal side. A comparison of ED50's for inhibition of single channels by DHPs added to the external side and ED50's for displacement of [3H]PN200-110 bound to the DHP receptor, revealed a good quantitative agreement. However, internal ED50's of channels were consistently higher than radioligand binding affinities by up to two orders of magnitude. Evidently, Ca channels of skeletal muscle are functionally coupled to two DHP receptor sites on opposite sides of the membrane.

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 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.

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