Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification

Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.     

Sodium and calcium currents in dispersed mammalian septal neurons

Voltage-gated Na+ and Ca2+ conductances of freshly dissociated septal neurons were studied in the whole-cell configuration of the patch-clamp technique. All cells exhibited a large Na+ current with characteristic fast activation and inactivation time courses. Half-time to peak current at -20 mV was 0.44 +/- 0.18 ms and maximal activation of Na+ conductance occurred at 0 mV or more positive membrane potentials. The average value was 91 +/- 32 nS (approximately 11 mS cm-2). At all membrane voltages inactivation was well fitted by a single exponential that had a time constant of 0.44 +/- 0.09 ms at 0 mV. Recovery from inactivation was complete in approximately 900 ms at -80 mV but in only 50 ms at -120 mV. The decay of Na+ tail currents had a single time constant that at -80 mV was faster than 100 microseconds. Depolarization of septal neurons also elicited a Ca2+ current that peaked in approximately 6-8 ms. Maximal peak Ca2+ current was obtained at 20 mV, and with 10 mM external Ca2+ the amplitude was 0.35 +/- 0.22 nA. During a maintained depolarization this current partially inactivated in the course of 200-300 ms. The Ca2+ current was due to the activity of two types of conductances with different deactivation kinetics. At -80 mV the closing time constants of slow (SD) and fast (FD) deactivating channels were, respectively, 1.99 +/- 0.2 and 0.11 +/- 0.03 ms (25 degrees C). The two kinds of channels also differed in their activation voltage, inactivation time course, slope of the conductance-voltage curve, and resistance to intracellular dialysis. The proportion of SD and FD channels varied from cell to cell, which may explain the differential electrophysiological responses of intracellularly recorded septal neurons.     

Time- and voltage-dependent components of Kv4.3 inactivation

Kv4.3 inactivation is a complex multiexponential process, which can occur from both closed and open states. The fast component of inactivation is modulated by the N-terminus, but the mechanisms mediating the other components of inactivation are controversial. We studied inactivation of Kv4.3 expressed in Xenopus laevis oocytes, using the two-electrode voltage-clamp technique. Inactivation during 2000 ms pulses at potentials positive to the activation threshold was described by three exponents (46 +/- 3, 152 +/- 13, and 930 +/- 50 ms at +50 mV, n = 7) whereas closed-state inactivation (at potentials below threshold) was described by two exponents (1079 +/- 119 and 3719 +/- 307 ms at -40 mV, n = 9). The fast component of open-state inactivation was dominant at potentials positive to -20 mV. Negative to -30 mV, the intermediate and slow components dominated inactivation. Inactivation properties were dependent on pulse duration. Recovery from inactivation was strongly dependent on voltage and pulse duration. We developed an 11-state Markov model of Kv4.3 gating that incorporated a direct transition from the open-inactivated state to the closed-inactivated state. Simulations with this model reproduced open- and closed-state inactivation, isochronal inactivation relationships, and reopening currents. Our data suggest that inactivation can proceed primarily from the open state and that multiple inactivation components can be identified.     

Voltage clamp study of fast excitatory synaptic currents in bullfrog sympathetic ganglion cells

Excitatory postsynaptic currents (EPSCs) have been studied in voltage- clamped bullfrog sympathetic ganglion B cells. The EPSC was small, rose to a peak within 1-3 ms, and then decayed exponentially over most of its time-course. For 36 cells at --50 mV (21-23 degrees C), peak EPSC size was --6.5 +/- 3.5 nA (mean +/- SD), and the mean decay time constant tau was 5.3 +/- 0.9 ms. tau showed a small negative voltage dependence, which appeared independent of temperature, over the range -- 90 to --30 mV; the coefficient of voltage dependence was --0.0039 +/- 0.0014 mV-1 (n = 29). The peak current-voltage relationship was linear between --120 and --30 mV but often deviated from linearity at more positive potentials. The reversal potential determined by interpolation was approximately --5 mV. EPSC decay tau had a Q10 = 3. The commonly used cholinesterase inhibitors, neostigmine and physostigmine, exhibited complex actions at the ganglia. Neostigmine (1 X 10(-5)M) produced a time-dependent slowing of EPSC decay without consistent change in EPSC size. In addition, the decay phase often deviated from a single exponential function, although it retained its negative voltage dependence. With 1 x 10(-6) M physostigmine, EPSC decay was slowed by the decay phase remained exponential. At higher concentrations of physostigmine, EPSC decay was markedly prolonged and was composed of at least two decay components. High concentrations of atropine (10(-5) to 10(-4) M) produced complex alterations in EPSC decay, creating two or more exponential components; one decay component was faster and the other was slower than that observed in untreated cells. These results suggest that the time-course of ganglionic EPSC decay is primarily determined by the kinetics of the receptor-channel complex rather than hydrolysis or diffusion of transmitter away from the postsynaptic receptors.     

Voltage-gated transient currents in bovine adrenal fasciculata cells. II. A-type K+ current

In whole cell patch clamp recordings on enzymatically dissociated adrenal zona fasciculata (AZF) cells, a rapidly inactivating A-type K+ current was observed in each of more than 150 cells. Activation of IA was steeply voltage dependent and could be described by a Boltzmann function raised to an integer power of 4, with a midpoint of -28.3 mV. Using the "limiting logarithmic potential sensitivity," the single channel gating charge was estimated to be 7.2 e. Voltage-dependent inactivation could also be described by a Boltzmann function with a midpoint of -58.7 mV and a slope factor of 5.92 mV. Gating kinetics of IA included both voltage-dependent and -independent transitions in pathways between closed, open, and inactivated states. IA activated with voltage-dependent sigmoidal kinetics that could be fit with an n4h formalism. The activation time constant, tau a, reached a voltage- independent minimum at potentials positive to 0 mV. IA currents inactivated with two time constants that were voltage independent at potentials ranging from -30 to +45 mV. At +20 mV, tau i(fast) and tau i(slow) were 13.16 +/- 0.64 and 62.26 +/- 5.35 ms (n = 34), respectively. In some cells, IA inactivation kinetics slowed dramatically after many minutes of whole cell recording. Once activated by depolarization, IA channels returned to the closed state along pathways with two voltage-dependent time constants which were 0.208 s, tau rec-f and 10.02 s, tau rec-s at -80 mV. Approximately 90% of IA current recovered with slow kinetics at potentials between -60 and -100 mV. IA was blocked by 4-aminopyridine (IC50 = 629 microM) through a mechanism that was strongly promoted by channel activation. Divalent and trivalent cations including Ni2+ and La3+ also blocked IA with IC50's of 467 and 26.4 microM, respectively. With respect to biophysical properties and pharmacology, IA in AZF cells resembles to some extent transient K+ currents in neurons and muscle, where they function to regulate action potential frequency and duration. The function of this prominent current in steroid hormone secretion by endocrine cells that may not generate action potentials is not yet clear.     

Spontaneous and GABA-induced single channel currents in cultured murine spinal cord neurons

Intracellular and patch clamp recordings were made from embryonic mouse spinal cord neurons growing in primary cell culture. Outside-out membrane patches obtained from these cells usually showed spontaneous single channel currents when studied at the resting potential (-56 +/- 1.5 mV). In 18 out of 30 patches tested, spontaneous single channel activity was abolished by making Tris+ the major cation on both sides of the membrane. The remaining patches continued to display spontaneous single channel currents under these conditions. These events reversed polarity at a patch potential of 0 mV and displayed a mean single channel conductance of 24 +/- 1.2 pS. Application of the putative inhibitory transmitter gamma-aminobutyric acid (0.5-10 microM) to outside-out patches of spinal cord cell membrane induced single channel currents in 10 out of 15 patches tested. These channels had a primary conductance of 29 +/- 2.8 pS in symmetrical 145 mM Cl- solutions. Frequency distributions for the open times of these channels were well fit by the sum of a fast exponential term ("of") with a time constant tau of = 4 +/- 1.3 ms and a slow exponential term ("os") with a time constant tau os = 24 +/- 8.1 ms. Frequency distributions for channel closed times were also well fit by a double exponential equation, with time constants tau cf = 2 +/- 0.2 ms and tau cs = 62 +/- 20.9 ms.     

State-dependent inactivation of the alpha1G T-type calcium channel.

We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the alpha1G channel, with symmetrical Na(+)(i) and Na(+)(o) and 2 mM Ca(2+)(o). After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential -100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from -120 to -70 mV (e-fold for 31 mV; tau = 2.5 ms at -100 mV), but tau = 12-17 ms from-40 to +60 mV. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100-300 ms, inactivation was strong but incomplete (approximately 98%). Inactivation was also produced by long, weak depolarizations (tau = 220 ms at -80 mV; V(1/2) = -82 mV), which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast (tau = 85 ms at -100 mV), but weakly voltage dependent. Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at -100 mV during recovery from inactivation, consistent with 相似文献   

Inactivation of L-type Ca channels in embryonic chick ventricle cells: dependence on the cytoskeletal agents colchicine and taxol.

This article shows that colchicine and taxol strongly influence the kinetics of L-type Ca channels in intact cardiac cells, and it suggests a mechanism for this action. It is known that colchicine disassociates microtubules into tubulin, and that taxol stabilizes microtubules. We have found that colchicine increases the probability that Ca channels are in the closed state and that taxol increases the probability they are in the open state. Moreover, taxol lengthens the mean open time of Ca channels. In this regard, taxol is similar to Bay-K 8644; however, Bay K works on inside-out patches, but taxol does not. Neither colchicine nor taxol alters the number of Ca channels in a patch. We have quantified these results as follows. It is known that L-type channels in embryonic chick heart ventricle cells have voltage- and current-dependent inactivation. In 10 mM Ba, channel conductance is linear in the range -10 to 20 mV. The conductance is 12 +/- 1 pS, and the extrapolated reversal potential is 42 +/- 2 mV (n = 3). In cell-attached patches, inactivation depends on the number of channels. One channel (holding at -80 mV and stepping to 0 mV for 500 ms) shows virtually no inactivation. However, three channels inactivate with a time constant of 360 +/- 20 ms (n = 6). In similar patches, colchicine (80 microM for 15 min) decreases the inactivation time constant to 162 +/- 33 ms (n = 4) and taxol (50 microM for 10 min) virtually abolishes inactivation (time constant 812 +/- 265 ms (n = 4)).(ABSTRACT TRUNCATED AT 250 WORDS)     

Low molecular weight poly(A)+ mRNA species encode factors that modulate gating of a non-Shaker A-type K+ channel

Voltage-dependent K+ channels control repolarization of action potentials and help establish firing patterns in nerve cells. To determine the nature and role of molecular components that modulate K+ channel function in vivo, we coinjected Xenopus oocytes with cRNA encoding a cloned subthreshold A-type K+ channel (mShal1, also referred to as mKv4.1) and a low molecular weight (LMW) fraction (2-4 kb) of poly(A)+ mRNA (both from rodent brain). Coinjected oocytes exhibited a significant (fourfold) increase in the surface expression of mShal1 K+ channels with no change in the open-channel conductance. Coexpression also modified the gating kinetics of mShal1 current in several respects. Macroscopic inactivation of whole oocyte currents was fitted with the sum of two exponential components. Both fast and slow time constants of inactivation were accelerated at all membrane potentials in coinjected oocytes (tau f = 47.2 ms vs 56.5 ms at 0 mV and tau s = 157 ms vs 225 ms at 0 mV), and the corresponding ratios of amplitude terms were shifted toward domination by the fast component (Af/As = 2.71 vs 1.17 at 0 mV). Macroscopic activation was characterized in terms of the time-to-peak current, and it was found to be more rapid at all membrane potentials in coinjected oocytes (9.9 ms vs 13.5 ms at 0 mV). Coexpression also leads to more rapid recovery from inactivation (approximately 2.4-fold faster at -100 mV). The coexpressed K+ currents in oocytes resemble currents expressed in mouse fibroblasts (NIH3T3) transfected only with mShal1 cDNA. These results indicate that mammalian regulatory subunits or enzymes encoded by LMW mRNA species, which are apparently missing or expressed at low levels in Xenopus oocytes, may modulate gating in some native subthreshold A-type K+ channels.     

Acute thyroid hormone promotes slow inactivation of sodium current in neonatal cardiac myocytes.

Sodium current (INa) inactivation kinetics in neonatal cardiac myocytes were analyzed using whole cell voltage clamp before and after acute treatments with thyroid hormone (3,5,3'-triiodo-L-thyronine, T3). In untreated neonatal myocytes, INa inactivation was predominantly mono-exponential, with 93 +/- 3% (S.D.; n = 9) of the peak amplitude decaying with a time constant, tau h1, of 1.8 +/- 0.5 ms at -30 mV. The remaining 7% of control INa decayed more slowly, with a time constant, tau h2, of 9.3 +/- 3.0 ms at -30 mV. The contribution of slowly-inactivating channels to peak current was increased from 7% to 43 +/- 27% within 5 min of exposure to 5-20 nM T3 (nine cells; P less than 0.005). The time constants for both the fast- and slow-inactivating components of peak current (tau h1 and tau h2) were not significantly changed by acute T3 treatment, nor was steady-state INa inactivation (h infinity) affected. Thyroid hormone action on sodium inactivation was partially reversible by lidocaine. These findings indicate that T3 acts at the neonatal cardiac cell membrane to promote slow inactivation kinetics in sodium channels.     

Voltage-gated transient currents in bovine adrenal fasciculata cells. I. T-type Ca2+ current

The whole cell version of the patch clamp technique was used to identify and characterize voltage-gated Ca2+ channels in enzymatically dissociated bovine adrenal zona fasciculata (AZF) cells. The great majority of cells (84 of 86) expressed only low voltage-activated, rapidly inactivating Ca2+ current with properties of T-type Ca2+ current described in other cells. Voltage-dependent activation of this current was fit by a Boltzmann function raised to an integer power of 4 with a midpoint at -17 mV. Independent estimates of the single channel gating charge obtained from the activation curve and using the "limiting logarithmic potential sensitivity" were 8.1 and 6.8 elementary charges, respectively. Inactivation was a steep function of voltage with a v1/2 of -49.9 mV and a slope factor K of 3.73 mV. The expression of a single Ca2+ channel subtype by AZF cells allowed the voltage-dependent gating and kinetic properties of T current to be studied over a wide range of potentials. Analysis of the gating kinetics of this Ca2+ current indicate that T channel activation, inactivation, deactivation (closing), and reactivation (recovery from inactivation) each include voltage-independent transitions that become rate limiting at extreme voltages. Ca2+ current activated with voltage- dependent sigmoidal kinetics that were described by an m4 model. The activation time constant varied exponentially at test potentials between -30 and +10 mV, approaching a voltage-independent minimum of 1.6 ms. The inactivation time constant (tau i) also decreased exponentially to a minimum of 18.3 ms at potentials positive to 0 mV. T channel closing (deactivation) was faster at more negative voltages; the deactivation time constant (tau d) decreased from 8.14 +/- 0.7 to 0.48 +/- 0.1 ms at potentials between -40 and -150 mV. T channels inactivated by depolarization returned to the closed state along pathways that included two voltage-dependent time constants. tau rec-s ranged from 8.11 to 4.80 s when the recovery potential was varied from - 50 to -90 mV, while tau rec-f decreased from 1.01 to 0.372 s. At potentials negative to -70 mV, both time constants approached minimum values. The low voltage-activated Ca2+ current in AZF cells was blocked by the T channel selective antagonist Ni2+ with an IC50 of 20 microM. At similar concentrations, Ni2+ also blocked cortisol secretion stimulated by adrenocorticotropic hormone. Our results indicate that bovine AZF cells are distinctive among secretory cells in expressing primarily or exclusively T-type Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Delays in inactivation development and activation kinetics in myxicola giant axons

Na inactivation was studied in Myxicola (two-pulse procedure, 6-ms gap between conditioning and test pulses). Inactivation developed with an initial delay (range 130-817 microseconds) followed by a simple exponential decline (time constant tau c). Delays (deviations from a simple exponential) are seen only for brief conditioning pulses were gNa is slightly activated. Hodgkin-Huxley kinetics with series resistance, Rs, predict deviations from a simple exponential only for conditioning pulses that substantially activate gNa. Reducing INa fivefold (Tris substitution) had no effect on either tau c or delay. Delay in not generated by Rs or by contamination from activation development. The slowest time constant in Na tails is approximately 1 ms (Goldman and Hahin, 1978) and the gap was 6 ms. Shortening the gap to 2 ms had no effect on either tau c or delay. Delay is a true property of the channel. Delay decreased with more positive conditioning potentials, and also decreased approximately proportionally with time to peak gNa during the conditioning pulse, as expected for sequentially coupled activation and inactivation. In a few cases the difference between Na current values for brief conditioning pulses and the tau c exponential could be measured. Difference values decayed exponentially with time constant tau m. The inactivation time course is described by a model that assumes a process with the kinetics of gNa activation as a precursor to inactivation.     

The initial inward current in spherical clusters of chick embryonic heart cells

The rapid inward sodium current in spherical clusters of 11-d-old embryonic chick heart cells, ranging in size between 65 and 90 micron diameter, was studied using the two-microelectrode voltage-clamp technique. Using these preparations, it was possible to resolve the activation phase of the rapid inward current for potentials negative to -25 mV at 37 degrees C. The rapid inward current exhibited a voltage and time dependence similar to that observed in other excitable tissues. It was initiated at potential steps more positive than -45 mV. The magnitude of the current reached its maximum value at a potential of approximately -20 mV. The measured reversal potential was that predicted by the Nernst equation for sodium ions. The falling phase of the current followed a single exponential time-course with a time constant of inactivation, tau h, ranging between 2.14 ms at -40 mV and 0.18 ms at -5 mV. The time constant of inactivation, tau h, determined by a single voltage-step protocol was compared to the constant, tau c, determined by a double voltage-step protocol and no significant different between the two constants of inactivation was found. Furthermore, the time constants of inactivation and reactivation at the same potential in the same preparation were similar. The results of this study demonstrate that the sodium current of heart cells recorded at 37 degrees C can be described by Hodgkin-Huxley kinetics with speeds approximately four times faster than the squid giant axon at 15 degrees C.     

Effect of ajmaline on transient outward current in rat ventricular myocytes

The mechanism of ajmaline-induced inhibition of the transient outward current (I(to)) has been investigated in right ventricular myocytes of rat using the whole cell patch clamp technique. Ajmaline decreased the amplitude and the time integral of I(to) in a concentration-dependent, but frequency- and use-independent manner. In contrast to the single exponential time course of I(to)-inactivation in control conditions (tau(i) = 37.1 +/- 2.7 ms), the apparent inactivation was fitted by a sum of two exponentials under the effect of ajmaline with concentration-dependent fast and slow components (tau(f) = 11.7 +/- 0.8 ms, tau(s) = 57.6 +/- 2.7 ms at 10 micromol/l) suggesting block development primarily in the open channel state. An improved expression enabling to calculate the association and dissociation rate constants from the concentration dependence of tau(f) and tau(s) was derived and resulted in k(on) = 4.57 x 10(6) +/- 0.32 x 10(6) mol(-1).l.s(-1) and k(off) = 20.12 +/- 5.99 s(-1). The value of K(d) = 4.4 micromol/l calculated as k(off) / k(on) was considerably lower than IC(50) = 25.9 +/- 2.9 micromol/l evaluated from the concentration dependence of the integrals of I(to). Simulations on a simple model combining Hodgkin-Huxley type gating kinetics and drug-channel interaction entirely in open channel state agreed well with the experimental data including the difference between the K(d) and IC(50). According to the model, the fraction of blocked channels increases upon depolarization and declines if depolarization is prolonged. The repolarizing step induces recovery from block with time constant of 52 ms. We conclude that in the rat right ventricular myocytes, ajmaline is an open channel blocker with fast recovery from the block at resting voltage.     

Effects of reduced PO2 on rapid-drive-induced hyperpolarization of diastolic transmembrane potential in feline cardiac Purkinje strands

Using standard microelectrode techniques, we evaluated effects of diminished oxygen tension on the magnitude and time course of frequency dependent changes in maximum diastolic transmembrane potential (MDP) and on alteration of action potential duration (APD) in feline Purkinje fibers. MDP was recorded continuously during a control period (cycle length (CL) = 1000 ms), during a 5-min period of rapid drive (CL = 400 ms) and following return to pacing CL = 1000 ms. Rapid drive resulted in hyperpolarization of MDP from control value; and after return to pacing CL = 1000 ms. MDP gradually depolarized, eventually attaining a steady state value within +/- 0.5 mV of the control value. The difference between hyperpolarized MDP value and final steady-state value was designated VH, and the decline of MDP towards steady-state value approximated an exponential function (time constant = tau VH). Exposure to reduced PO2 (75 +/- 2.1 mmHg vs. control 473 +/- 39.1 mmHg) (1 mmHg = 133.322 Pa) resulted in reduction in the magnitude of VH (6.2 +/- 3.43 mV vs. 7.8 +/- 2.73 mV, mean +/- SD, p less than 0.005) and shortening of APD within 0-24 min, while measurable prolongation of tau VH (75 +/- 18.5 vs. 54 +/- 9.0 s, p less than 0.005) began at 25-49 min following onset of reduced PO2. These observations suggest that rate-related changes of MDP in cardiac tissues are oxygen dependent, and they support previously reported analagous observations in nerve which suggested that frequency dependent potential changes may in part reflect alterations of electrogenic Na-K pump activity.     

Uncoupling of gating charge movement and closure of the ion pore during recovery from inactivation in the Kv1.5 channel

Both wild-type (WT) and nonconducting W472F mutant (NCM) Kv1.5 channels are able to conduct Na(+) in their inactivated states when K(+) is absent. Replacement of K(+) with Na(+) or NMG(+) allows rapid and complete inactivation in both WT and W472F mutant channels upon depolarization, and on return to negative potentials, transition of inactivated channels to closed-inactivated states is the first step in the recovery of the channels from inactivation. The time constant for immobilized gating charge recovery at -100 mV was 11.1 +/- 0.4 ms (n = 10) and increased to 19.0 +/- 1.6 ms (n = 3) when NMG(+)(o) was replaced by Na(+)(o). However, the decay of the Na(+) tail currents through inactivated channels at -100 mV had a time constant of 129 +/- 26 ms (n = 18), much slower than the time required for gating charge recovery. Further experiments revealed that the voltage-dependence of gating charge recovery and of the decay of Na(+) tail currents did not match over a 60 mV range of repolarization potentials. A faster recovery of gating charge than pore closure was also observed in WT Kv1.5 channels. These results provide evidence that the recovery of the gating elements is uncoupled from that of the pore in Na(+)-conducting inactivated channels. The dissociation of the gating charge movements and the pore closure could also be observed in the presence of symmetrical Na(+) but not symmetrical Cs(+). This difference probably stems from the difference in the respective abilities of the two ions to limit inactivation to the P-type state or prevent it altogether.     

Na(+) current contribution to the diastolic depolarization in newborn rabbit SA node cells

Isolated newborn, but not adult, rabbit sinoatrial node (SAN) cells exhibit spontaneous activity that (unlike adult) are highly sensitive to the Na(+) current (I(Na)) blocker TTX. To investigate this TTX action on automaticity, cells were voltage clamped with ramp depolarizations mimicking the pacemaker phase of spontaneous cells (-60 to -20 mV, 35 mV/s). Ramps elicited a TTX-sensitive current in newborn (peak density 0.89 +/- 0.14 pA/pF, n = 24) but not adult (n = 5) cells. When depolarizing ramps were preceded by steplike depolarizations to mimic action potentials, ramp current decreased 54.6 +/- 8.0% (n = 3) but was not abolished. Additional experiments demonstrated that ramp current amplitude depended on the slope of the ramp and that TTX did not alter steady-state holding current at pacemaker potentials. This excluded a steady-state Na(+) window component and suggested a kinetic basis, which was investigated by measuring TTX-sensitive I(Na) during long step depolarizations. I(Na) exhibited a slow but complete inactivation time course at pacemaker voltages (tau = 33.9 +/- 3.9 ms at -50 mV), consistent with the rate-dependent ramp data. The data indicate that owing to slow inactivation of I(Na) at diastolic potentials, a small TTX-sensitive current flows during the diastolic depolarization in neonatal pacemaker myocytes.     

Two distinct modulatory effects on calcium channels in adult rat sensory neurons.

D-ala2-D-leu5-enkephalin (100 to 1000 nM) reduces HVA Ca2+ currents of approximately 60% in 92% of the adult rat sensory neurons tested. In 80% of the cells sensitive to enkephalin, the reduction in Ca2+ current amplitude was associated with a prolongation of the current activation that was relieved by means of conditioning pulses in a potential range only about 10 mV positive to the current activation range in control conditions. The time course of the current activation was fitted to a single exponential in control, (tau = 2.23 msec +/- 0.14 n = 38) and double exponential with enkephalin, (tau 1 = 2.18 msec +/- 0.25 and tau 2 = 9.6 msec +/- 1, test pulse to -10 mV, 22 degrees C). A strong conditioning depolarizing prepulse speeded up the activation time course, completely eliminating the slow, voltage-sensitive exponential component, but it was only partial effective in restoring the current amplitude to control values. The voltage-independent inhibitory component that was not relieved could be recovered only by washing out enkephalin. In the remaining 20% of the cells affected, enkephalin decreased Ca2+ current amplitude without prolongation of Ca2+ channel activation. In these cases the conditioning voltage pulse was not effective in relieving the inhibition that persisted also at strong positive test potentials, on the outward currents. The voltage-dependent inhibition occurred slowly after enkephalin superfusion (tau congruent to 12 sec), whereas the voltage-independent one developed about ten times more rapidly. Dopamine (100 microM) could also induce both voltage-dependent and independent modulations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic currents in dispersed chemoreceptor cells of the mammalian carotid body

Ionic currents of enzymatically dispersed type I and type II cells of the carotid body have been studied using the whole cell variant of the patch-clamp technique. Type II cells only have a tiny, slowly activating outward potassium current. By contrast, in every type I chemoreceptor cell studied we found (a) sodium, (b) calcium, and (c) potassium currents. (a) The sodium current has a fast activation time course and an activation threshold at approximately -40 mV. At all voltages inactivation follows a single exponential time course. The time constant of inactivation is 0.67 ms at 0 mV. Half steady state inactivation occurs at a membrane potential of approximately -50 mV. (b) The calcium current is almost totally abolished when most of the external calcium is replaced by magnesium. The activation threshold of this current is at approximately -40 mV and at 0 mV it reaches a peak amplitude in 6-8 ms. The calcium current inactivates very slowly and only decreases to 27% of the maximal value at the end of 300-ms pulses to 40 mV. The calcium current was about two times larger when barium ions were used as charge carriers instead of calcium ions. Barium ions also shifted 15-20 mV toward negative voltages the conductance vs. voltage curve. Deactivation kinetics of the calcium current follows a biphasic time course well fitted by the sum of two exponentials. At -80 mV the slow component has a time constant of 1.3 +/- 0.4 ms whereas the fast component, with an amplitude about 20 times larger than the slow component, has a time constant of 0.16 +/- 0.03 ms. These results suggest that type I cells have predominantly fast deactivating calcium channels. The slow component of the tails may represent the activity of a small population of slowly deactivating calcium channels, although other possibilities are considered. (c) Potassium current seems to be mainly due to the activity of voltage-dependent potassium channels, but a small percentage of calcium-activated channels may also exist. This current activates slowly, reaches a peak amplitude in 5-10 ms, and thereafter slowly inactivates. Inactivation is almost complete in 250-300 ms. The potassium current is reversibly blocked by tetraethylammonium. Under current-clamp conditions type I cells can spontaneously fire large action potentials. These results indicate that type I cells are excitable and have a variety of ionic conductances. We suggest a possible participation of these conductances in chemoreception.