Separation of sodium and calcium channels in the surface membrane of molluscan nerve cells

By intracellular dialysis of isolated neurons of the mollusksHelix pomatia andLimnaea stagnalis and by a voltage clamp technique the characteristics of transmembrane ionic currents were studied during controlled changes in the ionic composition of the extracellular and intracellular medium. By replacing the intracellular potassium ions by Tris ions, functional blocking of the outward potassium currents was achieved and the inward current distinguished in a pure form. Replacement of Ringer's solution in the extracellular medium with sodium-free or calcium-free solution enabled the inward current to be separated into two additive components, one carried by sodium ions, the other by calcium ions. Sodium and calcium inward currents were found to have different kinetics and different potential-dependence: _mNa=1±0.5 msec, _mCa=3±1 msec, _hNa=8±2 msec, _hCa=115±10 msec (V_m=0), G_Na=0.5 (V_m=–21±2 mV), G_Ca=0.5 (V_m=–8±2 mV). Both currents remained unchanged by tetrodotoxin, but the calcium current was specifically blocked by cadmium ions (2·10^–3 M), verapamil, and D=600, and also by fluorine ions if injected intracellularly. All these results are regarded as evidence that the soma membrane of the neurons tested possesses separate systems of sodium and calcium ion-conducting channels. Quantitative differences are observed in the relative importance of the systems of sodium and calcium channels in different species of mollusks.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 183–191, March–April, 1976.     

Inactivation of calcium currents in the soma of spinal ganglia by removing the membrane potential depolarization shift

Kinetic and voltage-dependent characteristics of the inactivating action of incoming calcium currents were investigated in the somatic membrane of rat spinal ganglia neurons using an intracellular dialysis technique. It was shown that the "tail" of low-threshold calcium current could be reliably described by one exponent with a time constant of =1.2–1.8 msec at a repolarization potential of –90 mV. The "tail" of the high-threshold calcium current represented the sum of several exponents. The time constant of the main component which expressed inactivation of the high threshold calcium current was ^h=250–350 µsec. It was also shown that and ^h remained virtually unchanged for repolarization potentials in the subthreshold region; they increase, however, if the repolarizing potential is close to those potentials at which the corresponding component of calcium current is initially activated. A dependence was observed between the levels and ^h and duration of the depolarizing shift. Findings are discussed in the context of a three-tier model of calcium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 682–691, September–October, 1985.     

Effects of divalent cations on toad end-plate channels

Summary Miniature end-plate currents (MEPCs) and acetylcholine-induced current fluctuations were recorded in voltageclamped, glycerol-treated toad sartorius muscle fibers in control solution and in solutions with added divalent cations. In isosmotic solutions containing 20mm Ca or Mg, MEPCs had time constants of decay ( _D ) which were about 30% slower than normal. In isotonic Ca solutions (Na-free), greater increases in both _D and channel lifetime were seen; the null potential was –34 mV, and single-channel conductance decreased to approximately 5 pS. Zn or Ni, at concentrations of 0.1–5mm, were much more effective in increasing _D than Ca or Mg, although they did not greatly affect channel conductance. The normal temperature and voltage sensitivity of was not significantly altered by any of the added divalent cations. Surface potential shifts arising from screening of membrane fixed charge by divalent cations cannot entirely explain the observed increases in , especially when taken together with changes in channel conductance.     

Inactivation of voltage-dependent calcium current in an insulinoma cell line

We have studied the mechanism of Ca current inactivation in the -cell line HIT-T15 by conventional and perforated patch recording techniques, using two pulse voltage protocols and a combination of current and tail current measurements. In 5 mM Ca, from a holding potential of - 80 mV, the maximum current showed a complex time course of inactivation: a relatively fast, double exponential inactivation (_h1 12 ms and _h2 60 ms) and a very slowly inactivating component ( > 1 s). The faster component (_h1) was due to the voltage-dependent inactivation of a low-threshold-activated (LVA), T-type current, which deactivates more slowly ( 3–5 ms) than the other components ( 0.2–0.3 ms). The intermediate component (_h2) was due to the Ca-dependent inactivation of a portion of the high-threshold-activated (HVA) current. A saturating dose of the dihydropyridine (DHP) nifedipine (10 M) did not affect the LVA current, but inhibited by 68 ± 5% the transient, Ca-sensitive portion of the HVA current and by 33 ± 12% the long lasting component. We suggest that three components of the calcium current can be resolved in HIT cells and the main target of DHPs is a HVA current, which inactivates faster than the DHP-resistant HVA component and does so primarily through calcium influx. Correspondence to: C. Marchetti     

Three classes of potassium channels in large monopolar cells of the blowfly Calliphora vicina

Summary We have used single electrode voltage clamp in the intact animal and whole-cell recording from dissociated cell bodies to investigate the properties of potassium conductances in large monopolar cells (LMCs) of the first optic ganglion of the blowfly Calliphora vicina. Two classes of voltage gated potassium conductances were found: a delayed rectifier current (Kd) with slow inactivation (_inac = 1–3 sec), and an A current (Ka) showing both faster inactivation (_inac = 21 ms) and also more rapid activation. The reversal potential of both currents is ca. -90 mV with 2 mM [K_o] and 140 mM [K_i], and follows the Nernst slope with increasing [K_o]. The voltage operating range of Ka is unusually negative, with the mid point of the steady-state inactivation curve (V₅₀) at- 101 mV. V₅₀ for Kd is - 84 mV. Although no inward currents were detected, for technical reasons their presence cannot be excluded.In inside-out patches from LMC soma membranes the single channels underlying the currents both have a conductance of ca. 20 pS in symmetrical 140 mM K solutions and channel densities may be as high as 10/m². Less frequently, inside-out patches contained a large conductance (110 pS) calcium-activated potassium channel which existed almost exclusively in a rapidly flickering mode. Open probability increased with depolarization and Ca concentrations greater than 40 nM.In whole-cell recordings, dissociated LMC cell bodies fall into two classes with respect to their voltage sensitive currents: 37 % of cells only showed Kd; the remainder (63%) were dominated by Ka with a variable (0–30%) contribution from Kd. In the intact animal, intracellular recordings from LMCs, combined with dye-marking, indicate that cells expressing only Kd are type L3 cells, whilst L1 and L2 express predominantly Ka. Since L1 and L2 have resting potentials of ca. - 40 mV and maximum hyperpolarizations reaching -90 mV only transiently, inactivation of Ka is unlikely to be removed under most physiological conditions. In contrast, L3 cells have a more negative resting potential (–60 mV) and Kd should play a significant role in signal-shaping, in particular contributing to the falling phase of a prominent spike-like transient in response to dimming.Abbreviations Ka A current - Kd delayed rectifier - LMC large monopolar cell - L1-L3 classes thereof - TTX tetrodotoxin     

Effects of voltage and temperature changes on postsynaptic potentiation at the frog neuromuscular junction

The difference in the decay time constants of multiquantal endplate currents (EPC) produced by presenting paired stimuli 100 msec apart was measured during experiments on transversely cut neuromuscular preparations of the frog sartorius muscle. When acetylcholinesterase was inhibited by 3×10^–6 M prostigmine, decay time of the 2nd EPC (₂) was 39±8% longer than that of the first (₁) due to postsynaptic potentiation. It was found that degree of potentiation was not affected by membrane potential level within the –30 to –120 mV range. Several effects were produced by a drop in temperature: an increase in EPC decay time constant and in that of miniature endplate currents (MEPC) in particular, a slight drop in MEPC amplitude, and a reduction in EPC quantal content. By comparing paired EPC of equal quantal content at different temperatures it was found that potentiation was more pronounced at 12°C than at 22°C and the temperature coefficient Q₁₀ at which ₂ exceeds ₁ was 2.0±0.2 (n=7). The processes determining postsynaptic potential are clearly not voltage-dependent but have a complex dependence on temperature. Quantal content of EPC falls with reduced temperature, thereby restraining potentiation, while helping to retain residual transmitter activity.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Kurashov Medical Institute, Kazan'. Translated from Neirofiziologiya, Vol. 18, No. 4, pp. 512–518, July–August, 1986.     

Voltage dependence of calcium current deactivation in the somatic membrane of mouse sensory neurons

Voltage dependence of the deactivation kinetics of calcium inward currents was investigated in the somatic membrane of murine spinal ganglia neurons. It was found that deactivation of high threshold calcium current has a slower component (=0.80–0.85 msec at a repolarizing potential of –80 mV) as well as the principal transient exponential component (130 sec at the same potential repolarizing level). A dissimilar relationship exists between amplitudes of the transient and slower exponential components, describing deactivation of high threshold calcium current and degree of activation of the depolarizing shift in membrane potential; the former dependence is expressed by a sigmoid and the latter by a V-shaped curve. The slower component of deactivation of high threshold current was inhibited substantially by perfusing the cell with a Tris-PO₄-containing solution. Low-threshold calcium tail current undergoes slower deactivation (=1.1–1.2 msec) at a repolarizing potential of –160 mV. A relationship between the time constant of low threshold current deactivation and the type of penetrating cation used was observed. A kinetic model of calcium current deactivation is suggested, taking account of the three different types of calcium channels, (one low and two high threshold) present in the somatic membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 185–193, March–April, 1988.     

End plate currents at the physiological level of quantal secretion and after potentiation of transmitter release by 4-aminopyridine

The role of postysynaptic potentiation (PSP) and asynchronous secretion of acetylcholine (ACh) in the generation of multiquantal currents and end plate currents (EPC) was investigated under voltage clamp conditions in transected sartorius muscle of the frog before and after 4-aminopyridine (4-AP) treatment. Compared with miniature EPC (MEPC), showing an average quantum content of 249, multiquantal EPC has a larger amplitude, longer rise-time, and longer decay-time (_epc). Magnesium ions (6–10 mM) reduce the amplitude and _spec of EPC without affecting its rise-time. Rhythmical stimulation (10 Hz for 60 sec) results in reduced amplitude and but increased rise-time of EPC. D-turbocurarine (5×10^–7 M) and -bungarotoxin (1×10^–5 gm/ml) diminishes the difference between _epc and _mepc. In the presence of 4-AP, all these effects are much more pronounced. It is proposed that asynchronous secretion of ACh from motor nerve teminals causes prolongation of the rise-time and reduction of the amplitude of EPC but has little or no effect upon the decay rate of EPC. The slow decay of multiquantal EPC, both in the absence and in the presence of 4-AP, is almost entirely due to postynaptic interaction of ACh quanta, i.e., PSP.Kurashov Meidcal Institute, Kazhan. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 48–56, January–February, 1991.     

Postsynaptic currents in phasic and tonic muscle fibers of the rat extraocular muscle

Focal extracellular recordings were made of postjunctional currents produced at synapses of the inferior rectus eye muscle fibers by the spontaneous release of quanta of transmitter. These consisted of miniature endplate currents, or MEPC, in phasic fibers and miniature postjunctional currents, or MPJC, in tonic fibers. Open time of ionic channels (_chan) was also registered. In tonic fibers, MPJC lasted considerably longer than MEPC did in phasic fibers: rising time, decay time, and _chan in the former measured respectively 2.5, 4–5, and 2.2 times higher than in the latter. Acetylcholinesterace (AChE) inhibition produced a much greater (4.4-fold extension of current decay in phasic than in tonic fibers, where a 1.8-fold increase was seen, thereby reducing the gap between the decay time of currents in these fibers to a difference of 1.6 times. The more protracted decay of MPJC in tonic fibers compared with MEPC in phasic fibers is determined by the lower functional activity of AChE as well as the higher value of _chan. Duration of MEPC and magnitude of _chan in the "slow" phasic fibers of rat skeletal muscles fell well below the same parameters measured in the tonic fibers of the ocular muscle.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 120–129, January–February, 1987.     

Chemically modified cardiac Na+ channels and their sensitivity to antiarrhythmics: Is there a hidden drug receptor?

Elementary Na⁺ currents were recorded at 19°C in inside-out patches from cultured neonatal rat cardiocytes. In analyzing the sensitivity of chemically modified Na⁺ channels to several class 1 antiarrhythmic drugs, the hypothesis was tested that removal of Na⁺ inactivation may be accompanied by a distinct responsiveness to these drugs, open channel blockade.Iodate-modified and trypsin-modified cardiac Na⁺ channels are noninactivating but strikingly differ from each other by their open state kinetics, a O₁–O₂ reaction (_open(1) 1.4±0.3 msec; _open(2) 5.4±1.1 msec; at –40 mV) in the former and a single open state (_{open 3.0±0.5} msec; at –40 mV) in the latter. Lidocaine (150 mol/liter) like propafenone (10 mol/liter), diprafenone (10 mol/liter) and quinidine (20 mol/liter) in cytoplasmic concentrations effective to depress NP _o significantly can interact with both types of noninactivating Na⁺ channels to reduce the dwell time in the conducting configuration. lodate-modified Na⁺ channels became drug sensitive during the O₂ state. At –40 mV, for example, lidocaine reduced _open(2) to 62±5% of the control without detectable changes in _open(1). No evidence could be obtained that these inhibitory molecules would flicker-block the open Na⁺ pore. Drug-induced shortening of the open state, thus, is indicative for a distinct mode of drug action, namely interference with the gating process. Lidocaine proved less effective to reduce _open(2) when compared with the action of diprafenone. Both drugs apparently interacted with individual association rate constants, a_lidocaine was 0.64×10⁶ mol^–1 sec^–1 and a_diprafenone 13.6×10⁶ mol^–1 sec^–1. Trypsin-modified Na⁺ channels also appear capable of discriminating among these antiarrhythmics, the ratio a_diprafenone/a_lidocaine even exceeded the value in iodate-modified Na⁺ channels. Obviously, this antiarrhythmic drug interaction with chemically modified Na⁺ channels is receptor mediated: drug occupation of such a hypothetical hidden receptor that is not available in normal Na⁺ channels may facilitate the exit from the open state.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ko 778/2-4), Bonn.     

Permeance of Cs+ and Rb+ through the inwardly rectifying K+ channel in guinea pig ventricular myocytes

Summary Inward currents carried by external Cs, Rb, NH₄ and K through theI _K1 channel were studied using a whole-cell voltage clamp technique. Cs, NH₄, and Rb currents could be recorded negative to –40 mV following depolarizing prepulses (0 mV and 200–1000 msec in duration). The current activation displayed an instantaneous component followed by a monoexponential increase () to a peak amplitude. Subsequent inactivation was fit by a single exponential, _i. With hyperpolarization, and _i decreasede-fold per 36 and 25 mV, respectively. In Ca-free external solutions (pipette [Mg]0.3mm), inactivation was absent, consistent with the hypothesis that inactivation represents time- and voltage-dependent block of Cs, NH₄, and Rb currents by external Ca. The inactivation and degree of steady-state block was greatest when Cs was the charge carrier, followed by NH₄, and then Rb. K currents, however, did not inactivate in the presence of Ca. Na and Li did not carry any significant current within the resolution of our recordings. Comparison ofpeak inward current ratios (I _x/I_K) as an index of permeability revealed a higher permeance of Cs (0.15), NH₄ (0.30), and Rb (0.51) relative to K (1.0) than that obtained by comparing thesteady-state current ratios (CsNH₄RbK0.010.060.211.0). At any given potential, was smaller the more permeant the cation. In the absence of depolarizing prepulses, the amplitude of was reduced. Divalent-free solutions did not significantly affect activatio in the presence of 0.3mm pipette [Mg]. When pipette [Mg] was buffered to 50 m, however, removal of external Ca and Mg lead to a four- to fivefold increase in Cs currents and loss of both time-dependent activation and inactivation (reversible upon repletion of external Ca).These results suggest that (i) permeability ratios forI _K1 should account for differences in the degree to which monovalent currents are blocked by extracellular Ca and (ii) extracellular or intracellular divalent cations contribute to the slow phase of activation which may represent either (a) the actual rate of Mg or Ca extrusion from the channel into the cell, a process which may be enhanced by repulsive interaction with the incoming permeant monovalent cation or (b) an intrinsic gating process that is strongly modulated by the permeant monovalent ion and divalent cations.     

Responsiveness of cardiac Na+ channels to antiarrhythmic drugs: The role of inactivation

Summary Elementary Na⁺ currents were recorded at 9°C in inside-out patches from cultured neonatal rat heart myocytes. In characterizing the sensitivity of cooled, slowly inactivating cardiac Na⁺ channels to several antiarrhythmic drugs including propafenone, lidocaine and quinidine, the study aimed to define the role of Na⁺ inactivation for open channel blockade.In concentrations (1–10 mol/liter) effective to depressNP _o significantly, propafenone completely failed to influence the open state of slowly inactivating Na⁺ channels. With 1 mol/liter, _open changed insignificantly to 96±7% of the control. Even a small number of ultralong openings of 6 msec or longer exceeding _open of the whole ensemble several-fold and attaining _open (at –45 mV) in cooled, (-)-DPI-modified, noninactivating Na⁺ channels proved to be drug resistant and could not be flicker-blocked by 10 mol/liter propafenone. The same drug concentration induced in(-)-DPI-modified Na⁺ channels a discrete block with association and dissociation rate constants of 16.1 ± 5.3 × 10⁶ mol^–1 sec^–1 and 675 ± 25 sec^–1, respectively. Quinidine, known to have a considerable affinity for activated Na⁺ channels, in lower concentrations (5 mol/liter) left _open unchanged or reduced, in higher concentrations (10 mol/liter) _open only slightly to 81% of the predrug value whereasNP _o declined to 30%, but repetitive blocking events during the conducting state could never be observed. Basically the same drug resistance of the open state was seen in cardiac Na⁺ channels whose open-state kinetics had been modulated by the cytoplasmic presence of F^– ions. But in this case, propafenone reduced reopening and selectively abolished a long-lasting open state. This drug action is unlikely related to the inhibitory effect onNP _o since hyperpolarization and the accompanying block attenuation did not restore the channel kinetics. It is concluded that cardiac Na⁺ channels cannot be flicker-blocked by antiarrhythmic drugs unless Na⁺ inactivation is removed.     

Induced diploid gynogenesis and polyploidy in the ornamental (koi) carp Cyprinus carpio L.

The results of a series of experiments conducted in our laboratory on the ornamental common carp (koi), aimed at optimizing heat-shock chromosome-set manipulation procedures, are described. The timing of heat-shock initiation was expressed in the relative unit of embryological age (₀) in order to standardize this parameter, the absolute time for heat-shock initiation being calculated from duration of one ₀ at two different pre-treatment water temperatures. Heat shocks were applied within the periods of 0.05–0.60 ₀ and 1.20–2.20 ₀ which, respectively, cover the successive phases of the 2nd meiotic division and the 1st cleavage. The highest production of diploid gynogenetic offspring was observed when heat shocks were initiated at 0.15–0.25 ₀ and at 1.5 ₀, after insemination, corresponding to anaphase of meiosis-II, and metaphase of the 1st cleavage, respectively. Similar results were obtained irrespective of the different pre-treatment water temperatures, thus confirming the possibility of standardizing heat-shock timing by ₀.     

Evidence for two K+ currents activated upon hyperpolarization ofParamecium tetraurelia

Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K⁺ solutions elicits a complex of Ca²⁺ and K⁺ currents. The tail current that accompanies a return to holding potential (–40 mV) contains two K⁺ components. The tail current elicited by a step to –110 mV of 50-msec duration contains fast-decaying (3.5 msec) and slow-decaying (20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K⁺], suggesting that they represent pure K⁺ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K⁺ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca²⁺ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K⁺ currents are inhibited by extracellular TEA⁺ in a concentration-dependent, noncooperative manner, whereas the fast K⁺ current alone is inhibited by 0.7mm quinidine.     

Sodium channel opening as a precursor to inactivation

The time constant of the process producing the delay in Na inactivation development as determined by the two pulse method (_delay) was extracted and compared to that of the slowest Na activation process ₃ for the I _Na during the conditioning pulse of that same determination. _delay and two pulse inactivation _c values were computer generated using a nonlinear least squares algorithm. _h and single pulse inactivation _h values were independently generated for each determination also with the aid of the computer using the same non-linear least squares algorithm. In one determination at 2 mV, _c was 4.68 and _delay 0.494 ms while _h was 4.70 and ₃ 0.491 ms for a _c/_h of 0.996 and a _delay/₃ of 1.006. Mean _delay/₃ from five determinations in four axons, both Cs and K perfused, and spanning a potential range of-27 to 2mV was 1.068. The precursor process to inactivation is channel opening. Some fraction of channels presumably inactivate via another route where prior channel opening is not required.     

Some effects of short-chain phospholipids and n-alkanes on a transient potassium current (I A ) in identified Helix neurons

Many effects of short-chain phospholipids and n-alkanes on the squid axon sodium current (I _Na) are consistent with mechanisms involving changes in membrane thickness. Here, we suggest that the actions of short-chain phospholipids on an A-type potassium current (I _A ) in two-microelectrode voltage clamped Helix D1 and F77 neurons are incompatible with such simple mechanisms. Diheptanoyl phosphatidylcholine (diC₇PC, 0.2 and 0.3 mm) caused substantial (58 and 79%), and in some cases partially reversible, increases in I _A amplitude. These were correlated with hyperpolarizing shifts of up to –7 mV in the voltage dependence of current activation. The voltage dependence of steady-state inactivation was also moved in the hyperpolarizing direction. These effects are the opposite of those described for squid I _Na. 0.5 Saturated n-pentane and saturated n-hexane caused significant (–3 and –6 mV) hyperpolarizing shifts in the voltage dependence of I _A inactivation, qualitatively consistent with their effects on squid I _Na, while the voltage dependence of activation was moved slightly to the left or unchanged. Hydrocarbons had variable effects on peak current amplitude, although saturated n-pentane produced a clear suppression. DiC₇PC caused a 25% increase in the time constant of macroscopic I _A inactivation ( _b ) but 0.5 saturated n-pentane and saturated n-hexane reduced _b by 40%. The effects of these agents on current-clamped cells were broadly consistent with their opposing actions on _b —phospholipids tended to reduce excitability and n-alkanes tended to increase it. Possible mechanisms of I _A perturbation are discussed.We gratefully acknowledge financial support from the Science and Engineering Research Council and the Wellcome Trust. We would also like to thank Prof. H. Meves, Dr. N. Franks and Dr. W. Lieb for helpful discussions.     

Effect of axoplasmic transport blockade on end-plate currents in frog muscle fibers

Evoked and spontaneous end-plate currents (EPC) were studied in normal voltage-clamped frog sartorius muscle fibers and 2 weeks after application of colchicine to the nerve innervating the muscle to block axoplasmic transport in its fibers. Application of colchicine was found to reduce the rate of rise and to prolong decay of EPC without affecting the amplitude of the EPC and miniature EPC, the quantum composition of EPC, and the frequency of miniature EPC. The histogram of distribution of the time constant () of EPC decay under normal conditions follows the normal law, but after application of colchicine to the nerve it is shifted to the right, with separation of two modes (₁ and ₂). Three types of synapses can be distinguished from the character of EPC decay: monoexponential decay with ₁ (44%), biexponential decay with ₁ and ₂ (39%), and monoexponential decay with ₂ (19%). An increase in of EPC decay is accompanied by strengthening of the dependence of this process on the clamping voltage. The current-voltage characteristic and reversal potential of EPC are unchanged. It is suggested that the change in character of EPC decay after application of colchicine to the motor nerve is due to the appearance of acetylcholine-activated ionic channels in the muscle membrane with a longer duration of the open state and with potential-dependence of the open state similar to that taking place after muscle denervation.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 17, No. 2, pp. 204–211, March–April, 1985.     

Multiple-channel conductance states and voltage regulation of embryonic chick cardiac gap junctions

Summary We used the double whole-cell voltage-clamp technique on ventricle cell pairs isolated from 7-day chick heart to measure the conductance of their gap junctions (G _j) and junctional channels ( _j) with a steady-state voltage difference (V _j) applied across the junction. Currents were recorded from single gap junction channels (i _j) as symmetrical rectangular signals of equal size and opposite sign in the two cells, and _j was measured from i _j/V _j. We observed channel openings at six reproducible conductance levels with means of 42.6, 80.7, 119.6, 157.7, 200.4 and 240.3 pS. More than half of all openings were to the 80-and 160-pS conductance levels. The probability that a high conductance event (e.g., 160 or 240 pS) results from the random simultaneous opening of several 40-pS channels is small, based on their frequency of occurrence and on the prevalence of shifts between small and large conductance states with no intervening 40-pS steps. Our results are consistent with three models of embryonic cardiac gap junction channel configuration: a homogeneous population of 40-pS channels that can open cooperatively in groups of up to six; a single population of large channels with a maximal conductance near 240 pS and five smaller substates; or several different channel types, each with its own conductance. G _j was determined from the junctional current (I _j) elicited by rectangular pulses of applied transjunctional voltage as I _j/V _j. It was highest near 0 V _j and was progressively reduced by application of V _j between 20 and 80 mV or –20 and –80 mV. In response to increases in V _j, G _j decayed in a voltage-and timedependent fashion. After a 6-sec holding period at 0 V _j, the initial conductance (G _init) measured immediately after the onset of an 80-mV step in V _j was nearly the same as that measured by a 10-mV prepulse. However, during 6-sec pulses of V _j>±20 mV, G _j declined over several seconds from G _init to a steady-state value (G _ss). At potentials greater than ±20 mV the current decay could be fit with biexponential curves with the slow decay time constant ( ₂) 5–20 times longer than ₁. For the response to a step to 80 mV V _j, for example, ₁=127 msec and ₂=2.6 sec. The rate of current decay in response to smaller positive or negative steps in V _j was slower, the magnitude of the decline was smaller, and the ratio ₂/ ₁ decreased. The relationship between G _init and V _j was approximately linear between 0 and 80 mV or –80 mV. whereas the relationship between G _ss and V _j was nonlinear beyond ±20 mV. Upon returning to 0 V _j, G _j recovered with a biexponential time course, reaching its maximal value after several seconds; recovery time constants after a step in V _j from 80 to 0 mV were 225 msec and 1.9 sec. In the resting state, at low junctional voltage, high conductance channel activity (160–240 pS) is favored. Voltage-dependent decline of G _j results in part from a shift from high to lower conductance states.We thank Ms. B.J. Duke for technical assistance and for preparation of the cell cultures and Drs. L.J. DeFelice and D. Eaton for stimulating and helpful discussions of the results.     

Analysis of the T-type calcium channel in embryonic chick ventricular myocytes

Summary T-type calcium channels (I _T channels) were studied in cell-attached patch electrode recordings from the ventricular cell membrane of 14-day embryonic chick heart. All experiments were performed in the absence of Ca²⁺ with Na⁺ (120mm) as the charge carrier.I _T channels were distinguished from L-type calcium channels (I _L) by their more negative activation and inactivation potential ranges; their smaller unitary slope conductance (26 pS), and their insensitivity to isoproterenol or D600. Inactivation kinetics were voltage dependent. The time constant of inactivation was 37 msec when the membrane potential was depolarized 40 mV from rest (R+40 mV), and 20 msec atR+60 mV. The frequency histogram of channel open times ₀ was fit by a single-exponential curve while that of closed times _c was biexponeintial. _o was the same atR+40 mV andR+60 mV whereas _c was shortened atR+60 mV. The open-state probability (P _o) increased with depolarization: 0.35 atR+40 mV, 0.8 atR+60 mV and 0.88 atR+80 mV. This increase inP _o at depolarized potentials could be accounted for by the decrease in _c.     

Effects of arachidonic acid and the other long-chain fatty acids on the membrane currents in the squid giant axon

Summary The effects of arachidonic acid and some other long-chain fatty acids on the ionic currents of the voltage-clamped squid giant axon were investigated using intracellular application of the test substances. The effects of these acids, which are usually insoluble in solution, were examined by using -cyclodextrin as a solvent. -cyclodextrin itself had no effect on the excitable membrane. Arachidonic acid mainly suppresses the Na current but has little effect on the K current. These effects are completely reversed after washing with control solution. The concentration required to suppress the peak inward current by 50% (ED₅₀) was 0.18mm, which was 10 times larger than that of medium-chain fatty acids like 2-decenoic acid. The Hill number was 1.5 for arachidonic acid, which is almost the same value as for medium-chain fatty acids. This means that the mechanisms of the inhibition are similar in both long- and medium-chain fatty acids. When the long-chain fatty acids were compared, the efficacy of suppression of Na current was about the same value for arachidonic acid, docosatetraenoic acid and docosahexaenoic acid. The suppression effects of linoleic acid and linolenic acid on Na currents were one-third of that of arachidonic acid. Oleic acid had a small suppression effect and stearic acid had almost no effect on the Na current. The currents were fitted to equations similar to those proposed by Hodgkin and Huxley (Hodgkin, A.L., Huxley, A.F. (1952)J. Physiol (London) 117:500–544) and the change in the parameters of these equations in the presence of fatty acids were calculated. The curve of the steady-state activation parameter (m ) for the Na current against membrane potential and the time constant of activation ( _m ) were shifted 10 mV in a depolarizing direction by the application of fatty acids. The time constant for inactivation ( _h ) has almost unaffected by application of these fatty acids.