scholarly journals Voltage-dependent modulation of Ca channel current in heart cells by Bay K8644.

1986 ◽  
Vol 88 (3) ◽  
pp. 369-392 ◽  
Author(s):  
M C Sanguinetti ◽  
D S Krafte ◽  
R S Kass
Keyword(s):  
Rate Constant ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Heart Cells ◽  
Ca Channel ◽  
Drug Induced ◽  
Constant Change ◽  
Voltage Dependent ◽  
Simple Kinetic Model ◽  
And Shifts

We have investigated the voltage-dependent effects of the dihydropyridine Bay K8644 on Ca channel currents in calf Purkinje fibers and enzymatically dispersed rat ventricular myocytes. Bay K8644 increases the apparent rate of inactivation of these currents, measured during depolarizing voltage pulses, and shifts both channel activation and inactivation in the hyperpolarizing direction. Consequently, currents measured after hyperpolarizing conditioning pulses are larger in the presence of drug compared with control conditions, but are smaller than control if they are measured after positive conditioning pulses. Most of our experimental observations on macroscopic currents can be explained by a single drug-induced change in one rate constant of a simple kinetic model. The rate constant change is consistent with results obtained by others with single channel recordings.

Characterization of a newly found stretch-activated KCa,ATP channel in cultured chick ventricular myocytes

1999 ◽  
Vol 276 (6) ◽  
pp. H1827-H1838 ◽  
Author(s):  
Takashi Kawakubo ◽  
Keiji Naruse ◽  
Tatsuaki Matsubara ◽  
Nigishi Hotta ◽  
Masahiro Sokabe
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Heart Cells ◽  
Patch Clamp Technique ◽  
Selective Channel ◽  
New Type ◽  
Inside Out ◽  
Ion Selectivities ◽  
Channel Type

With the use of the patch-clamp technique, five kinds of stretch-activated (SA) ion channels were identified on the basis of their single-channel conductances and ion selectivities in cultured chick ventricular myocytes. Because a high-conductance K+-selective channel predominated among these channels, we concentrated on characterizing its properties mostly using excised inside-out patches. With 145 mM KCl solution in the pipette and the bath, the channel had a conductance of 199.8 ± 8.2 pS ( n = 22). The ion selectivities among K+, Na+, Ca2+, and Cl− as estimated from their permeability ratios were P Na/ P K= 0.03, P Ca/ P K= 0.025, and P Cl/ P K= 0.026. The probability of the channel being open (Po) increased with the Ca2+concentration in the bath ([Ca2+]b; dissociation constant K d = 0.51 μM at +30 mV) and membrane potential (voltage at half-maximal Po= 39.4 mV at 0.35 μM [Ca2+]b). The channel was blocked by gadolinium, tetraethylammonium, and charybdotoxin from the extracellular surface and, consequently, was identified as a Ca2+-activated K+(KCa) channel type. The channel was also reversibly activated by ATP applied to the intracellular surface ( K d = 0.74 mM at 0.10 μM [Ca2+]bat +30 mV). From these data taken together, we concluded that the channel is a new type of KCachannel that could be designated as an “SA KCa,ATP channel.” To our knowledge, this is the first report of KCa channel in heart cells.

Prepulse-induced mode 2 gating behavior with and without β-adrenergic stimulation in cardiac L-type Ca channels

1999 ◽  
Vol 276 (6) ◽  
pp. C1338-C1345 ◽  
Author(s):  
Yuji Hirano ◽  
Takashi Yoshinaga ◽  
Mitsushige Murata ◽  
Masayasu Hiraoka
Keyword(s):  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Ca Channel ◽  
Ca Channels ◽  
Adrenergic Stimulation ◽  
Open Probability ◽  
Mode 2 ◽  
Voltage Dependent ◽  
Phosphorylation Mechanism ◽  
Prepulse Facilitation

Mode 2 gating of L-type Ca channels is characterized by high channel open probability ( NP o) and long openings. In cardiac myocytes, this mode is evoked physiologically in two apparently different circumstances: membrane depolarization (prepulse facilitation) and activation of protein kinase A. To examine whether the phosphorylation mechanism is involved during prepulse-induced facilitation of cardiac L-type Ca channels, we used isolated guinea pig ventricular myocytes to analyze depolarization-induced modal gating behavior under different basal levels of phosphorylation. In control, NP o measured at 0 mV was augmented as the duration of prepulse to +100 mV was prolonged from 50 to 400 ms. This was due to the induction of mode 2 gating behavior clustered at the beginning of test pulses. Analysis of open time distribution revealed that the prepulse evoked an extra component, the time constant of which is not dependent on prepulse duration. When isoproterenol (1 μM) was applied to keep Ca channels at an enhanced level of phosphorylation, basal NP o without prepulse was increased by a factor of 3.6 ± 2.2 ( n = 6). Under these conditions, prepulse further increased NP o by promoting long openings with the same kinetics of transition to mode 2 gating (τ ≅ 200 ms at +100 mV). Likewise, recovery from mode 2 gating, as estimated by the decay of averaged unitary current, was not affected after β-stimulation (τ ≅ 25 ms at 0 mV). The kinetic behavior independent from the basal level of phosphorylation or activity of cAMP-dependent protein kinase suggests that prepulse facilitation of the cardiac Ca channel involves a mechanism directly related to voltage-dependent conformational change rather than voltage-dependent phosphorylation.

scholarly journals A PIP2 substitute mediates voltage sensor-pore coupling in KCNQ activation

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Yongfeng Liu ◽  
Xianjin Xu ◽  
Junyuan Gao ◽  
Moawiah M. Naffaa ◽  
Hongwu Liang ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Membrane Lipids ◽  
Voltage Sensor ◽  
Structural Basis ◽  
Drug Induced ◽  
Kcnq Channels ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Action Potential Prolongation ◽  
Voltage Sensor Domain

AbstractKCNQ family K+ channels (KCNQ1-5) in the heart, nerve, epithelium and ear require phosphatidylinositol 4,5-bisphosphate (PIP2) for voltage dependent activation. While membrane lipids are known to regulate voltage sensor domain (VSD) activation and pore opening in voltage dependent gating, PIP2 was found to interact with KCNQ1 and mediate VSD-pore coupling. Here, we show that a compound CP1, identified in silico based on the structures of both KCNQ1 and PIP2, can substitute for PIP2 to mediate VSD-pore coupling. Both PIP2 and CP1 interact with residues amongst a cluster of amino acids critical for VSD-pore coupling. CP1 alters KCNQ channel function due to different interactions with KCNQ compared with PIP2. We also found that CP1 returned drug-induced action potential prolongation in ventricular myocytes to normal durations. These results reveal the structural basis of PIP2 regulation of KCNQ channels and indicate a potential approach for the development of anti-arrhythmic therapy.

scholarly journals Permeation in the dihydropyridine-sensitive calcium channel. Multi-ion occupancy but no anomalous mole-fraction effect between Ba2+ and Ca2+.

1990 ◽  
Vol 95 (5) ◽  
pp. 911-939 ◽  
Author(s):  
D T Yue ◽  
E Marban
Keyword(s):  
Mole Fraction ◽  
Single Channel ◽  
Total Concentration ◽  
Debye Length ◽  
Heart Cells ◽  
Ca Channel ◽  
Ion Permeation ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Independent Evidence

We investigated the mechanism whereby ions cross dihydropyridine-sensitive (L-type) Ca channels in guinea pig ventricular myocytes. At the single-channel level, we found no evidence of an anomalous mole-fraction effect like that reported previously for whole-cell currents in mixtures of Ba and Ca. With the total concentration of Ba + Ca kept constant at 10 (or 110) mM, neither conductance nor absolute unitary current exhibits a paradoxical decrease when Ba and Ca are mixed, thereby weakening the evidence for a multi-ion permeation scheme. We therefore sought independent evidence to support or reject the multi-ion nature of the L-type Ca channel by measuring conductance at various permeant ion concentrations. Contrary to the predictions of models with only one binding site in the permeation pathway, single-channel conductance does not follow Michaelis-Menten kinetics as Ba activity is increased over three orders of magnitude. Two-fold variation in the Debye length of permeant ion solutions has little effect on conductance, making it unlikely that local surface charge effects could account for these results. Instead, the marked deviation from Michaelis-Menten behavior was best explained by supposing that the permeation pathway contains three or more binding sites that can be occupied simultaneously. The presence of three sites helps explain both a continued rise in conductance as [Ba2+] is increased above 110 mM, and the high single-channel conductance (approximately 7 pS) with 1 mM [Ba2+] as the charge carrier; the latter feature enables the L-type channel to carry surprisingly large currents at physiological divalent cation concentrations. Thus, despite the absence of an anomalous mole-fraction effect between Ba and Ca, we suggest that the L-type Ca channel in heart cells supports ion flux by a single-file, multi-ion permeation mechanism.

scholarly journals Kinetics of 9-aminoacridine block of single Na channels.

1984 ◽  
Vol 84 (3) ◽  
pp. 361-377 ◽  
Author(s):  
D Yamamoto ◽  
J Z Yeh
Keyword(s):  
Rate Constant ◽  
Voltage Dependence ◽  
Single Channel ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Voltage Dependent ◽  
The Mean ◽  
Kinetics Of

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.

scholarly journals Effects of magnesium on inactivation of the voltage-gated calcium current in cardiac myocytes.

1989 ◽  
Vol 94 (4) ◽  
pp. 745-767 ◽  
Author(s):  
H C Hartzell ◽  
R E White
Keyword(s):  
Steady State ◽  
Ventricular Myocytes ◽  
Internal Surface ◽  
State Level ◽  
Inactivation Kinetics ◽  
Ca Channel ◽  
Patch Clamp Technique ◽  
Inactivation Curve ◽  
Current Voltage ◽  
Voltage Dependent

The effects of changes in intracellular and extracellular free ionized [Mg2+] on inactivation of ICa and IBa in isolated ventricular myocytes of the frog were investigated using the whole-cell configuration of the patch-clamp technique. Intracellular [Mg2+] was varied by internal perfusion with solutions having different calculated free [Mg2+]. Increasing [Mg2+]i from 0.3 mM to 3.0 mM caused a 16% reduction in peak ICa amplitude and a 36% reduction in peak IBa amplitude, shifted the current-voltage relationship and the inactivation curve approximately 10 mV to the left, decreased relief from inactivation, and caused a dramatic increase in the rate of inactivation of IBa. The shifts in the current-voltage and inactivation curves were attributed to screening of internal surface charge by Mg2+. The increased rate of inactivation of IBa was due to an increase in both the steady-state level of inactivation as well as an increase in the rate of inactivation, as measured by two-pulse inactivation protocols. Increasing external [Mg2+] decreased IBa amplitude and shifted the current-voltage and inactivation curves to the right, but, in contrast to the effect of internal Mg2+, had little effect on the inactivation kinetics or the steady-state inactivation of IBa at potentials positive to 0 mV. These observations suggest that the Ca channel can be blocked quite rapidly by external Mg2+, whereas the block by [Mg2+]i is time and voltage dependent. We propose that inactivation of Ca channels can occur by both calcium-dependent and purely voltage-dependent mechanisms, and that a component of voltage-dependent inactivation can be modulated by changes in cytoplasmic Mg2+.

scholarly journals Na+ Occupancy and Mg2+ Block of the N-Methyl-d-Aspartate Receptor Channel

2001 ◽  
Vol 117 (3) ◽  
pp. 275-286 ◽  
Author(s):  
Yongling Zhu ◽  
Anthony Auerbach
Keyword(s):  
Membrane Potential ◽  
Dissociation Constant ◽  
Rate Constant ◽  
Single Channel ◽  
Dissociation Rate ◽  
Equilibrium Dissociation Constant ◽  
Voltage Dependent ◽  
Lock In Effect ◽  
Equilibrium Dissociation ◽  
Competing Ions

The effect of extracellular and intracellular Na+ on the single-channel kinetics of Mg2+ block was studied in recombinant NR1-NR2B NMDA receptor channels. Na+ prevents Mg2+ access to its blocking site by occupying two sites in the external portion of the permeation pathway. The occupancy of these sites by intracellular, but not extracellular, Na+ is voltage-dependent. In the absence of competing ions, Mg2+ binds rapidly (>108 M−1s−1, with no membrane potential) to a site that is located 0.60 through the electric field from the extracellular surface. Occupancy of one of the external sites by Na+ may be sufficient to prevent Mg2+ dissociation from the channel back to the extracellular compartment. With no membrane potential; and in the absence of competing ions, the Mg2+ dissociation rate constant is >10 times greater than the Mg2+ permeation rate constant, and the Mg2+ equilibrium dissociation constant is ∼12 μM. Physiological concentrations of extracellular Na+ reduce the Mg2+ association rate constant ∼40-fold but, because of the “lock-in” effect, reduce the Mg2+ equilibrium dissociation constant only ∼18-fold.

Asymmetric voltage dependence of embryonic cardiac gap junction channels

1996 ◽  
Vol 270 (1) ◽  
pp. C276-C285 ◽  
Author(s):  
Y. H. Chen ◽  
R. L. DeHaan
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Cardiac Development ◽  
Heart Cells ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Voltage Dependent ◽  
Chick Heart ◽  
Embryonic Chick Heart ◽  
Conductance States

The voltage dependence of junctional conductance (Gj) and the unitary channel behavior of junctions in most pairs of 3-day, 7-day, and 18-day embryonic chick heart cells are symmetrical, i.e., they are independent of the direction of polarization of junctional potential (Vj). With either cell depolarized relative to its neighbor, unitary channel events have a maximal unit conductance (yj) near 240 pS and five substates at nearly equal 40-pS increments down to near 40 pS (6, 9). Using the dual patch-clamp technique, we demonstrate here that, in a fraction of such cell pairs, Vj-dependent channel kinetics are asymmetric. Depolarization of one cell causes a larger and faster voltage-dependent decline in Gj than the same depolarization of the other cell. In a typical asymmetric preparation, depolarization of the strongly Vj-dependent side caused an immediate series of 47 +/- 16 pS closing steps in single-channel current (ij), followed by virtual cessation of channel activity. After depolarization of the less Vj-sensitive side, channel activity (56 +/- 13 pS) continues for many seconds. The large-conductance states (160-240 pS) observed in the electrically symmetric junctions were absent from the asymmetric preparations. In these cell pairs, connexin (Cx) 42, Cx43, and Cx45 could be immunolocalized at the junctional surfaces. We postulate that the asymmetry of voltage dependence in some cell pairs results from a preponderance of heterochannels formed from these different connexins. The frequency of asymmetric pairs obtained from 3-day, 7-day, and 18-day embryonic hearts was 50% (4/8), 24% (6/25), and 12.5% (1/8), suggesting that the fraction of heterochannels in the junctions decreases with cardiac development.

Citrate alters Ca channel gating and selectivity in rabbit ventricular myocytes

1992 ◽  
Vol 262 (1) ◽  
pp. C191-C198 ◽  
Author(s):  
L. V. Hryshko ◽  
D. M. Bers
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Monovalent Cation ◽  
Ca Channel ◽  
Channel Conductance ◽  
Na Current ◽  
Surface Charge Effect ◽  
Rabbit Ventricular Myocytes ◽  
Rabbit Ventricular Muscle

Addition of 10 mM citrate at constant free extracellular Ca concentration [( Ca]o; 2 mM) reduced contraction in rabbit ventricular muscle and isolated myocytes. We have recently shown that extracellular citrate decreases contraction and Ca current (ICa) in cardiac muscle by a direct effect on Ca channels rather than by Ca buffering per se [D. M. Bers, L. V. Hryshko, S. M. Harrison, and D. Dawson. Am. J. Physiol. 260 (Cell Physiol. 29): C900-C909, 1991]. Citrate rapidly depressed peak ICa and shifted both the peak ICa and the apparent reversal potential (Erev) to more negative potentials. When the impermeant cations, tetraethylammonium or N-methylglucamine were used instead of intracellular Cs, the citrate-induced shift in Erev was reduced or eliminated but depression of ICa was still observed. Thus citrate appears to alter the selectivity (PCa/PCs) of the Ca channel and reduce ICa. We also studied the effects of citrate on Na current through the Ca channel, observed when the divalent cation concentration is submicromolar. This current, termed INS for nonspecific, also exhibited leftward shifts in peak INS and smaller changes in Erev in the presence of citrate. However, neither peak INS nor single-channel conductance were affected by citrate. Thus the reduced PCa/PCs is due primarily to alteration of Ca permeation rather than monovalent cation permeation. Activation and inactivation curves for both ICa and INS were shifted toward more negative potentials by citrate. The shifts in gating and peak current to more negative membrane potentials would be consistent with a surface charge effect. The much larger shift in Erev for ICa (than for INS) is consistent with a reduction in Ca selectivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Single calcium channel currents of arterial smooth muscle at physiological calcium concentrations

1992 ◽  
Vol 263 (5) ◽  
pp. C948-C952 ◽  
Author(s):  
M. Gollasch ◽  
J. Hescheler ◽  
J. M. Quayle ◽  
J. B. Patlak ◽  
M. T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Single Channel ◽  
Membrane Potentials ◽  
Ca Channel ◽  
Ca Channels ◽  
Arterial Smooth Muscle ◽  
Voltage Dependent ◽  
High Concentrations ◽  
Single Channel Currents ◽  
Channel Currents

Entry of Ca through voltage-dependent Ca channels is an important regulator of the function of smooth muscle, cardiac muscle, and neurons. Although Ca channels have been extensively studied since the first descriptions of Ca action potentials (P. Fatt and B. Katz. J. Physiol. Lond. 120: 171-204, 1953), the permeation rate of Ca through single Ca channels has not been measured directly under physiological conditions. Instead, single Ca channels have typically been examined using high concentrations (80-110 mM) of another divalent charge carrier, Ba, so as to maximize the amplitude of the single-channel currents. Calculations of unitary currents at 2 mM Ca indicated that the single-channel currents would be immeasurably small (i.e., < 0.1 pA). We provide here the first direct measurements of single Ca channel currents at a physiological Ca concentration. Contrary to earlier estimates, we have found that currents through single Ca channels in arterial smooth muscle are 0.1-0.3 pA at 2 mM Ca and physiological membrane potentials. These relatively large unitary currents permit direct measurement of Ca channel properties under conditions that do not distort their function. Our data also indicate that Ca permeates these channels at relatively high rates in physiological Ca concentrations and membrane potentials.

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