Kinetics Of INa Research Articles

Kinetic analysis of cAMP-activated Na+ current in the molluscan neuron. A diffusion-reaction model.

cAMP-activated Na+ current (INa,cAMP) was studied in voltage-clamped neurons of the seaslug Pleurobranchaea californica. The current response to injected cAMP varied in both time course and amplitude as the tip of an intracellular injection electrode was moved from the periphery to the center of the neuron soma. The latency from injection to peak response was dependent on the amount of cAMP injected unless the electrode was centered within the cell. Decay of the INa,cAMP response was slowed by phosphodiesterase inhibition. These observations suggest that the kinetics of the INa,cAMP response are governed by cAMP diffusion and degradation. Phosphodiesterase inhibition induced a persistent inward current. At lower concentrations of inhibitor, INa,cAMP response amplitude increased as expected for decreased hydrolysis rate of injected cAMP. Higher inhibitor concentrations decreased INa,cAMP response amplitude, suggesting that inhibitor-induced increase in native cAMP increased basal INa,cAMP and thus caused partial saturation of the current. The Hill coefficient estimated from the plot of injected cAMP to INa,cAMP response amplitude was close to 1.0. An equation modeling INa,cAMP incorporated terms for diffusion and degradation. In it, the first-order rate constant of phosphodiesterase activity was taken as the rate constant of the exponential decay of the INa,cAMP response. The stoichiometry of INa,cAMP activation was inferred from the Hill coefficient as 1 cAMP/channel. The equation closely fitted the INa,cAMP response and simulated changes in the waveform of the response induced by phosphodiesterase inhibition. With modifications to accommodate asymmetric INa,cAMP activation, the equation also simulated effects of eccentric electrode position. The simple reaction-diffusion model of the kinetics of INa,cAMP may provide a useful conceptual framework within which to investigate the modulation of INa,cAMP by neuromodulators, intracellular regulatory factors, and pharmacological agents.

Sodium currents in smooth muscle cells freshly isolated from stomach fundus of the rat and ureter of the guinea-pig.

1. Inward currents elicited by depolarization from holding potentials of -80 to -10 mV in single smooth muscle cells isolated from stomach fundus of the rat and ureter of the guinea-pig had two components. The initial fast component (Ifi) was activated and mostly inactivated within 1-2 and 10 ms, respectively, at 21 degrees C. The following sustained component (Isi) lasted over 50 and 500 ms in fundus and ureter cells, respectively. Ifi was blocked by tetrodotoxin but not affected by 0.5 microM-mu-conotoxin in both types of cells. Isi was abolished by the substitution of extracellular Ca2+ with Mn2+. 2. The sensitivity of Ifis to TTX was markedly different in fundus and ureter cells. The half-inhibition was obtained at 870 and 11 nM, respectively. The amplitude of Ifi was highly dependent on extracellular Na+ concentration in a solution containing 2.2 mM-Mn2+ and 0 mM-Ca2+ in both cells. It is concluded that Ifis in these cells are TTX-sensitive and mu-conotoxin-insensitive Na+ currents. 3. Some of the kinetics of INa measured at 10 degrees C were markedly different in fundus and ureter cells. The current-voltage relationships for Ifi in fundus and ureter cells had peaks at about -10 and 0 mV, respectively. The voltage dependence of the steady-state inactivation of Ifi was also significantly different in these cell types. The half-inactivation voltages were about -74 and -45 mV, respectively. The recovery time course from inactivation in fundus cells was about 10 times slower than that in ureter at -80 mV, where it was 25 ms. 4. The contribution of Ifi to the rising phase of an action potential was examined using TTX under current clamp mode at 21 degrees C. A fast notch-like potential elicited by a subthreshold stimulus for action potential generation was blocked by TTX in both types of cells. Action potentials elicited by a stimulus around threshold were occasionally suppressed by TTX, whereas an action potential was never observed when extracellular Ca2+ was replaced with Mn2+. 5. In conclusion, the existence of at least two types of Na+ channel currents, which were distinguished by their TTX sensitivity and kinetics, was strongly suggested in smooth muscle cells from the rat fundus and the guinea-pig ureter. INa in these cells may have a physiological role to accelerate the generation of an action potential by triggering a rapid activation of ICa, while not being essential for activation of action potentials.

Acute effects of repetitive depolarization on sodium current in chick myocytes.

Acute effects of repetitive depolarization on the inward Na+ current (INa) of cultured embryonic chick atrial cells were studied using the whole cell patch-clamp technique. Stimulation rates of 1 Hz or greater produced a progressive decrement of peak INa. With depolarizations to 0 mV of 150-ms duration, applied at 2 Hz from a holding potential of -100 mV, the steady-state decrement was approximately 20%. The magnitude of this effect increased with stimulation frequency and with test potential depolarization and decreased with membrane hyperpolarization. Analysis of INa kinetics revealed that reactivation was sufficiently slow to preclude complete recovery from inactivation with interpulse intervals less than 1,000 ms. Moreover, reactivation accelerated markedly with membrane hyperpolarization, in parallel with the response to repetitive stimulation. The multiexponential time course of recovery of peak INa from repetitive depolarization was similar to that observed after single stimuli; however, there was a shift toward a greater proportion of current recovering with the slower of two time constants. It is concluded that incomplete recovery from inactivation is responsible for the decrement in INa observed with short interpulse intervals.

Na(+)‐activated K+ channels and voltage‐evoked ionic currents in brain stem and parasympathetic neurones of the chick.

1. Patch-clamp and computer-modelling techniques were used to study the activation of Na(+)-activated K+ channels (IK(Na] in dissociated neurones from the embryonic chick ciliary ganglion and the embryonic chick brain stem. 2. Numerical solutions of diffusion equations suggested that Na+ accumulation as a result of Na+ influx through voltage-sensitive Na+ channels (INa) is insufficient to allow for alteration in the gating of IK(Na) channels. 3. Whole-cell recordings using two independent micropipettes were made from chick ciliary-ganglion neurones. These showed that transient outward currents were present only when there were clear indications of incomplete voltage clamp. 4. Single-electrode whole-cell recordings from ciliary-ganglion neurones showed that transient tetrodotoxin (TTX)-sensitive outward currents were present, but only when partial TTX blockade produced significant alterations in the kinetics of INa. In cells that were properly voltage clamped, there was no effect of TTX on the kinetics of INa or on voltage-evoked outward currents. 5. Examination of the relationship between peak INa and the command potential showed that transient outward currents were only present in neurones that showed sharp deviations from the behaviour expected of a cell that is adequately voltage clamped. Transient outward currents were not present in cells that were adequately voltage clamped. 6. Application of TTX to isolated outside-out patches obtained from ciliary ganglion neurones eliminated voltage-evoked inward currents but had no effect on outward currents. 7. Isolated inside-out patches obtained from ciliary-ganglion neurones did not contain IK(Na) channels. These patches usually contained Ca(2+)-activated K+ channels (IK(Ca] with a unitary conductance of around 200 pS when [K+]o = 150 mM and [K+]i = 75 mM. 8. Two-electrode whole-cell recordings from cultured brain stem neurones showed that transient outward currents were present only when there were clear indications of incomplete voltage clamp. 9. Application of TTX caused blockade of inward but not outward currents in brain stem neurones voltage clamped with a single whole-cell pipette. TTX had no effect on the kinetics of INa. Application of TTX to outside-out patches isolated from the same cells blocked only the inward currents. 10. Isolated inside-out patches obtained from brain stem neurones contained IK(Na) channels that could be activated by exposure of the cytoplasmic face of the patch membrane to 75 mM-Na+. These channels had a predominant conductance state of around 100 pS when [K+]o = 150 mM and [K+]i = 75 mM. The IK(Na) channels were not activated by 1 mM-Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane currents of spiking cells isolated from turtle retina

We examined the membrane properties of spiking neurons isolated from the turtle (Pseudemys scripta) retina. The cells were maintained in culture for 1-7 days and were studied with the whole cell patch clamp technique. We utilized cells whose perikaryal diameters were greater than 15 microns since Kolb (1982) reported that ganglion cell perikarya in Pseudemys retina are 13-25 microns, whereas amacrine perikarya are less than 14 microns in diameter. We identified 5 currents in the studied cells: (1) a transient sodium current (INa) blocked by TTX, (2) a sustained calcium current (ICa) blocked by cobalt and enhanced by Bay-K 8644, (3) a calcium-dependent potassium current (IK(Ca)), (4) an A-type transient potassium current (IA) somewhat more sensitive to 4-AP than TEA, (5) a sustained potassium current (IK) more sensitive to TEA than 4-AP. The estimated average input resistance of the cells at -70 mV was 720 +/- 440 M omega. When all active currents were blocked, the membrane resistance between -130 and +20 mV was 2.5 G omega. When examined under current clamp, some cells produced multiple spikes to depolarizing steps of 0.1-0.3 nA, whereas other cells produced only a single spike irrespective of the strength of the current pulse. Most single spikers had an outward current that rose to a peak relatively slowly, whereas multiple spikers tend to have a more rapidly activating outward current. Under current clamp, 4-AP slowed the repolarization phase of the spike thus broadening it, but did not always abolish the ability to produce multiple spikes. TEA induced a depolarized plateau following the initial spike which precluded further spikes. It thus appears that the spiking patterns of the retinal cells are shaped primarily by the kinetics of INa, IK and IA and to a lesser extent by IK(Ca).

Development of the fast sodium current in early embryonic chick heart cells

Single ventricle cells were dissociated from the hearts of two-, three-, four- or seven-day-old chick embryos, and were maintained in vitro for an additional 6 to 28 hr. Rounded 13 to 18 micron cells with input capacitance of 5 to 10 pF were selected for analysis of fast sodium current (INa). Voltage command protocols designed to investigate the magnitude, voltage dependence, and kinetics of INa were applied with patch electrodes in the whole-cell clamp configuration. INa was present in over half of the 2d, and all 3d, 4d and 7d cells selected. The current showed no systematic differences in activation kinetics, voltage dependence, or tetrodotoxin (TTX) sensitivity with age or culture conditions. Between the 2d and 7d stages, the rate of current inactivation doubled and channel density increased about eightfold. At all stages tested, INa was blocked by TTX at a half-effective concentration of 0.5 to 1.0 nM. We conclude that the lack of Na dependence of the action potential upstroke on the second day of development results from the relatively depolarized level of the diastolic potential, and failure to activate the small available excitatory Na current. The change from Ca to Na dependence of the upstroke during the third to the seventh day of incubation results partly from the negative shift of the diastolic potential during this period, and in part from the increase in available Na conductance.

Site and mechanism of activation of proton‐induced sodium current in chick dorsal root ganglion neurones.

1. In dissociated and cultured 1- to 2-day-old chick dorsal root ganglion cells, and in isolated outside-out membrane patches, a large transient current lasting 1-2 s could be activated upon step increases in [H+]o. The proton-induced current reversed direction at the Na+ equilibrium potential, was abolished completely in the absence of Na+, and was therefore labelled INa(H). 2. To investigate the activation and deactivation kinetics of INa(H) at the single-channel level, we employed isolated membrane patches and a method whereby we could change the external solution in less than 1 ms. 3. In outside-out membrane patches, INa(H) was fully activated within 2 ms between pH 6.7 and 5.7. Half-times of activation decreased with increasing [H+]o. The calculated association rate constant was 9.5 x 10(9) M-1 s-1. 4. Deactivation of INa(H), following a step reduction in [H+]o, occurred with half-times of within 1.3-2 ms. 5. In the continued presence of an activating solution (pH 6.7 and 1 mM-Ca2+), INa(H) inactivated slowly, with a time constant of about 300 ms. 6. Inactivation showed a limited dependence on [Ca2+]o. The time constant of inactivation increased from about 300-500 ms as [Ca2+]o was decreased from 5 to 0.1 mM. Further decrease in [Ca2+]o did not significantly increase the time course of inactivation. Increases in [Ca2+]i from 10(-9) to 10(-3) M had no effect on the activation or inactivation kinetics of INa(H). 7. Conditioning proton concentrations which by themselves failed to activate any channel openings, partially inactivated INa(H). 8. Recovery from inactivation appeared to follow a time course similar to that of inactivation itself. 9. INa(H) could not be activated in inside-out patches. A step increase in proton concentration outside a cell-attached patch was also ineffective at producing INa(H) in the patch. Intracellular pH between 7.9 and 6.7 had no effect on the activation or inactivation of INa(H). 10. The activation and inactivation kinetics were not significantly voltage dependent. 11. The single-channel conductance associated with the activation of INa(H) was 28 pS in symmetrical 120 mM-NaCl solutions and remained constant throughout the time course of INa(H). 12. During activation of the voltage-gated calcium current, ICa, a step increase in proton concentration caused a rapid (ca. 2 ms) suppression of ICa which was more than that predicted from the steady-state effects of H+ on ICa. This effect was independent of [Na+] and the direction of INa(H).(ABSTRACT TRUNCATED AT 400 WORDS)

The sensitivity of apical Na+ permeability in frog skin to hypertonic stress.

Na+ transport across abdominal skins of the frog species Rana esculenta and Rana pipiens was analyzed by recording short-circuit current (Isc), transepithelial conductance (Gt), and the current noise generated by the random blockage of apical Na+ channels by the diuretic, amiloride. Specific Na+ current (INa) and conductance (GNa), as reflected by the amiloride-sensitive part of Isc and Gt, respectively, were markedly depressed after addition of some osmotically active substances, like sugars or alcohols to the mucosal Na+-Ringer solution. These hypertonicity-induced reactions were fast and fully reversible, even at mucosal osmolarities of 1 Osmol. With mucosal solutions of moderate hyperosmolarity a recovery of INa and GNa was observed in presence of the osmotic gradient. This "regulatory" current showed to be carried by Na+ through the Na+-specific apical channels. Contrary to the fast current drop during the initial phase of hyperosmotic shocks, the "osmoregulation" was considerably slower. The recovery of INa was only complete at smaller osmotic gradients but became more and more suppressed at higher osmolarities. Steady-state analysis of the kinetics of the Na+-specific current revealed that the current depression by osmotic shocks obeys Michaelis-Menten kinetics. This current depression at high osmolarities, as well as during the initial phase before "osmoregulation" with small osmotic gradients, can be described in terms of a non-competitive inhibition. This was also suggested by Na+-concentration jump experiments indicating a reduction of the maximal, apical Na+ permeability as mechanism of the hypertonicity-induced drop in INa. The INa kinetics after complete "osmoregulation" were, however, indistinguishable from the isotonic control condition.(ABSTRACT TRUNCATED AT 250 WORDS)

A.D.C. magnet power supply for 125 V, 500 A with controlled silicon rectifiers

Dispersion of action potential repolarization is known to be an important arrhythmogenic factor in cardiopathies such as Brugada syndrome. In this work, we analyze the effect of a variation in sodium current (INa) inactivation and a heterogeneous rise of transient outward current (Ito) in the probability of reentry in epicardial tissue. We use the Luo–Rudy model of epicardial ventricular action potential to study wave propagation in a one-dimensional fiber. Spatial dispersion in repolarization is introduced by splitting the fiber into zones with different strength of Ito. We then analyze the pro-arrhythmic effect of a variation in the relaxation time and steady-state of the sodium channel fast inactivating gate h. We quantify the probability of reentry measuring the percentage of reexcitations that occurs in 200 beats. We find that, for high stimulation rates, this percentage is negligible, but increases notably for pacing periods above 700 ms. Surprisingly, with decreasing INa inactivation time, the percentage of reexcitations does not grow monotonically, but presents vulnerable windows, separated by values of the INa inactivation speed-up where reexcitation does not occur. By increasing the strength of L-type calcium current ICaL above a certain threshold, reexcitation disappears. Finally, we show the formation of reentry in stimulated two-dimensional epicardial tissue with modified INa kinetics and Ito heterogeneity. Thus, we confirm that while Ito dispersion is necessary for phase-2 reentry, altered sodium inactivation kinetics influences the probability of reexcitation in a highly nonlinear fashion.