Role of Potassium Channels in Facilitation of Transmitter Release from Frog Motor Nerve Ending (Electrophysiology and Mathematical Simulation)

In experiments on neuromuscular junctions in the frog m. thoraco-cutaneous, we studied changes in the transmitter release and shape of the nerve ending (NE) response related to high-frequency (10 or 50 sec^-1) rhythmic stimulation of the motor nerve; an extracellular recording technique was used. At a low extracellular Ca²⁺ concentration, rhythmic stimulation resulted in a gradual increase in the quantum content of end-plate currents, i.e., in facilitation. Simultaneously, the third (positive) phase of the NE response became smaller, the amplitude of the second (negative) phase of this response also decreased, while the duration of this phase increased. Modifications of the NE response upon stimulation with a 10 sec^-1 frequency were more clearly expressed than those at 50 sec^-1 stimulation. In Ca²⁺-free solutions, rhythmic stimulation was accompanied by analogous modifications of the shape of NE responses, and the dynamics of these changes were the same at both the above-mentioned stimulation frequencies. When 0.5-1.0 mM tetraethylammonium was applied, 10 sec^-1 stimulation was accompanied by no facilitation of transmitter release; at 50 sec^-1 stimulation, this phenomenon was observed but was weaker than in the control, and the shape of NE responses underwent only mild changes. Simulation of electrogenesis in the studied structure showed that modifications of the NE response shape related to rhythmic 10 sec^-1 stimulation can develop in the case of a gradual decrease in the voltage-dependent potassium membrane conductivity, which results in prolongation of the de- and repolarization phases of action potentials and increases in the amplitude and duration of the inward calcium current. At higher stimulation frequencies (50 sec^-1), this mechanism is accompanied by a gradual increase in the Ca²⁺-dependent potassium conductivity, due to an increase in the intracellular Ca²⁺ concentration. These data allow us to conclude that the intensity of facilitation of transmitter release from the frog motor NE is related not only to accumulation of residual calcium, but also to changes of presynaptic calcium current due to modification of the kinetics of functioning of the potassium channels.     

Permeation and gating properties of the novel epithelial Ca(2+) channel

The recently cloned epithelial Ca(2+) channel (ECaC) constitutes the Ca(2+) influx pathway in 1,25-dihydroxyvitamin D(3)-responsive epithelia. We have combined patch-clamp analysis and fura-2 fluorescence microscopy to functionally characterize ECaC heterologously expressed in HEK293 cells. The intracellular Ca(2+) concentration in ECaC-expressing cells was closely correlated with the applied electrochemical Ca(2+) gradient, demonstrating the distinctive Ca(2+) permeability and constitutive activation of ECaC. Cells dialyzed with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid displayed large inward currents through ECaC in response to voltage ramps. The corresponding current-voltage relationship showed pronounced inward rectification. Currents evoked by voltage steps to potentials below -40 mV partially inactivated with a biexponential time course. This inactivation was less pronounced if Ba(2+) or Sr(2+) replaced Ca(2+) and was absent in Ca(2+)-free solutions. ECaC showed an anomalous mole fraction behavior. The permeability ratio P(Ca):P(Na) calculated from the reversal potential at 30 mM [Ca(2+)](o) was larger than 100. The divalent cation selectivity profile is Ca(2+) > Mn(2+) > Ba(2+) approximately Sr(2+). Repetitive stimulation of ECaC-expressing cells induced a decay of the current response, which was greatly reduced if Ca(2+) was replaced by Ba(2+) and was virtually abolished if [Ca(2+)](o) was lowered to 1 nM. In conclusion, ECaC is a Ca(2+) selective channel, exhibiting Ca(2+)-dependent autoregulatory mechanisms, including fast inactivation and slow down-regulation.     

Repetitive firing in motor nerve endings: Divalent cation,rhythmic activity,and cholinergic agent modulation

Repetitive firing (RF) as two or more end plate currents (EPC) induced by a single stimulus of the motor nerve was studied in sartorius frog muscle under voltage clamp conditions. In the presence of 4-aminopyridine (1.10^–4 M), RF as two EPC with 3–8 msec intervals was found in half of the cells (n=35). When calcium ion concentration was increased from 1.8 to 5.4 mM, magnesium to 5–9 mM, and rhythmic activity beginning with 0.05 Hz and above, RF stopped, but when strontium or barium was substituted for calcium, RF intensified. In the presence of barium, the repeated EPC quantity reached six-eight per one nerve impulse. Slow currents arose in some of the cells when disruption of the EPC series was stimulated. Proserine, an anticholinesterase agent, enhanced RF, whereas -bungarotoxin, a cholinoreceptor blocker, had no effect. The role that calcium and calcium-activated potassium currents play in generating and terminating mechanisms of RF, is discussed.S. V. Kurashov Medical Institute, Russian Federation Ministry of Public Health, Kazan. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 387–395, July–August, 1992.     

Correlation of Rates of Calcium Entry and Release of Endogenous Norepinephrine in Rat Brain Region Synaptosomes

Voltage-dependent 45Ca2+ uptake and endogenous norepinephrine (NE) release were measured simultaneously in synaptosomes isolated from rat hypothalamus, brainstem, and cerebellum at 1, 3, 5, 15, and 30 s. In synaptosomes depolarized by 125 mM KCl, 45Ca2+ uptake and NE release exhibited fast and slow components. Rates of NE release and 45Ca2+ uptake were fastest from 0 to 1 s. NE release and 45Ca2+ uptake rates from 1 to 5 s were less than 15% of 0-1 s rates. Both resting (5 mM KCl) and depolarization-induced (125 mM KCl) NE release paralleled 45Ca2+ uptake from 1 to 30 s. Voltage-dependent NE release was approximately 1% and 2% of total synaptosomal NE content at 1- and 30-s measurement intervals, respectively, and did not differ between the three brain regions studied. Calcium and potassium dependence studies showed that NE release was stimulated by increased potassium and that depolarization-induced NE release was dependent on the presence of external calcium. These results show that calcium-dependent NE release from synaptosomes is correlated with calcium entry. Both processes exhibit fast and slow temporal components.     

Facilitation and Depression of Neuromuscular Transmission under Conditions of Blockade of Ryanodine Receptors

In our experiments on mice, end-plate currents (EPC) evoked by stimulation of the phrenic nerve were intracellularly recorded in neuromuscular synaptic junctions of the phrenic muscle. We studied the effects of a specific blocker of ryanodine receptors, ryanodine (10 to 20 M), on the amplitude and time parameters of EPC under conditions of tetanic facilitation and depression of synaptic transmission at frequencies of stimulation of 4 to 200 sec^-1. Ryanodine inhibited facilitation at stimulation frequencies of 7 to 70 sec^-1 (with maximum effect at 20 sec^-1) and accelerated depression. In the presence of ryanodine, an initial rundown of the EPC amplitude in the course of depression of transmission increased at high frequencies of stimulation (50 to 100 sec^-1), whereas the EPC amplitude at the plateau level decreased already at low frequencies (4 to 7 sec^-1). We concluded that the changes in facilitation and depression resulted from blocking of the presynaptic ryanodine receptors by ryanodine. It seems probable that calcium release from the calcium stores in murine motor terminals is a factor involved in the control of processes of transmitter secretion during short-term rhythmic activation of the junction.     

The strontium-induced calcium-release process and its implication in contractility of skeletal muscle of Rana ridibunda

The electrical and mechanical activities of isolated frog muscle fibres have been recorded simultaneously under conditions (chloride-free saline containing 78.5 mM strontium acetate substituting for NaCl and CaCl2) that allow the development of a tubular strontium permeability. Under voltage-clamp conditions a large part of the contraction is due to the slow inward ISr since both are inhibited by Ni (10 mM). The remaining component of contraction, which seems to be potential-dependent, is not abolished by tetracaine (40 microM) which blocks the current-dependent component. A cumulative effect of strontium, which is not observed in the presence of Ni, leads to a 60-80% reduction in contractility for an estimated [Sr]i near 3 X 10(-4) M while the ending of the contraction observed when Sr is replaced by Ba is never obtained. In contrast no cumulative effect is observed when Ca substitutes for Sr. The first evoked inward current following a caffeine contracture fails to elicit a contraction, but in Ringer 78.5 Sr, contractility is progressively restored by successive depolarizations up to an amplitude which corresponds to 25-40% of the maximum activity. In the presence of Ca instead of Sr, the restoration of contractility reaches 100%. This recovery does not occur when the inward current is blocked by Ni. After strontium loading, a calcium entry fails immediately and reversibly to induce a mechanical response while barium ions induce a progressive and irreversible block of contractility. These results suggest that the strontium entry during successive depolarizations leads to a progressive replacement of intrareticular calcium by strontium. When all the calcium ions have been substituted for by strontium ions, the contractile apparatus remains capable of being activated by intrareticular strontium.     

Differential effects of Ba2+, Sr2+, and Ca2+ on stimulation-induced changes in transmitter release at the frog neuromuscular junction

Endplate potentials (EPP) were recorded from the frog sartorius neuromuscular junction under conditions of low quantal content to study the effect of Ba2+, Sr2+, and Ca2+ on the changes in evoked transmitter release that occur during and after repetitive stimulation. The addition of 0.1-1 mM Ba2+ or Sr2+ to the Ca2+-containing bathing solution, or the replacement of Ca2+ with 0.8-1.4 mM Sr2+, led to a greater increase in EPP amplitudes during and immediately after repetitive stimulation. These changes in release were analyzed in terms of the four apparent components of increased transmitter release that have previously been distinguished on the basis of their kinetic properties. The Ba2+-induced increase in EPP amplitudes was associated with an increase in the magnitude but not the time constant of decay of augmentation. Ba2+ had little effect on potentiation or the first and second components of facilitation. The Sr2+-induced increase in EPP amplitudes was associated with an increase in the magnitude and the time constant of decay of the second component of facilitation. Sr2+ had little effect on potentiation, augmentation, or the first component of facilitation. The selective effects of Ba2+ on augmentation and of Sr2+ on the second component of facilitation were reversible and could be obtained in the presence of the other ion. The addition of 0.1-0.3 mM Ca2+ to the bathing solution had little effect on potentiation, augmentation, or the two components of facilitation. These results provide pharmacological support for the proposal that there are four different components of increased transmitter release associated with repetitive stimulation and suggest that the underlying factors in the nerve terminal that give rise to these components can act somewhat independently of one another.     

The intracellular calcium and mechanisms of endocytosis of synaptic vesicles at the frog nerve ending

In the experiments on frog motor nerve endings of cutaneous pectoris muscle, made by extracellular recording of synaptic signals, it has been shown that the increase in intracellular calcium ion concentration in the nerve ending (by enhance of extracellular potassium ion concentration, or by addition of caffeine) leads to an increase in the miniature end-plate potential frequency, which is preserved over the whole period (about 10 min) of action of these substrates. The rhythmic stimulation of motor nerve (20 or 100 imp/s) quickly leads to a decrease in the end plate potentials amplitude. It has been shown by fluorescent microscopy with the use of endocytotic marker FM 1-43 that in the course of a short time exposition (5 min) in a high potassium solution (40 mM) or caffeine (5 mM), light spots appeared in the nerve ending. This shows that synaptic vesicles undergo intensive processes of endocytosis. During a longer exposition (30 min) no light spots were revealed, whereas the nerve ending width increased. This data allowed to propose that the process of endocytosis was blocked. In the presence of even lower concentrations of potassium ions and caffeine, and during a long rhythmic stimulation (20 or 100 imp/s) no blocking of endocytosis was revealed. It is concluded that high concentrations of intracellular calcium in the frog motor nerve ending leads to a reversible block of endocytosis, while exocytosis in synaptic vesicles is proceeding.     

Modulation of calcium channels by norepinephrine in internally dialyzed avian sensory neurons

Modulation of voltage-dependent Ca channels by norepinephrine (NE) was studied in chick dorsal root ganglion cells using the whole-cell configuration of the patch-clamp technique. Cells dialyzed with K+ and 2-10 mM EGTA exhibited Ca action potentials that were reversibly decreased in duration and amplitude by NE. Ca channel currents were isolated from other channel contributions by using: (a) tetrodotoxin (TTX) to block gNa, (b) internal K channel impermeant ions (Cs or Na/N-methylglucamine mixtures) as K substitutes, (c) external tetraethylammonium (TEA) to block K channels, (d) internal EGTA to reduce possible current contribution from Ca-activated channels. A marked decline (rundown) of Ca conductance was observed during continual dialysis, which obscured reversible NE effects. The addition of 2-5 mM MgATP to the intracellular solutions greatly retarded Ca channel rundown and permitted a clear assessment of modulatory drug effects. The inclusion of an intracellular creatine phosphate/creatine phosphokinase nucleotide regeneration system further stabilized Ca channels, which permitted recording of Ca currents for up to 3 h. NE reversibly decreased both steady state Ca currents and Ca tail currents in Cs/EGTA/MgATP-dialyzed cells. A possible role of several putative intracellular second messengers in NE receptor-Ca channel coupling was investigated. Cyclic AMP or cyclic GMP added to the intracellular solutions at concentrations several orders of magnitude higher than the Kd for activation of cyclic nucleotide-dependent protein kinases did not block or mask the expression of the NE-mediated decrease in gCa. Addition of internal EGTA to a final concentration of 10 mM also did not affect the expression of the NE response. These results suggest that neither cyclic AMP nor cyclic GMP nor Ca is acting as a second messenger coupling the NE receptor to the down-modulated Ca channel population.     

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.     

Nature of increase in quantal release by the thallous ion at frog end plates with and without nerve stimulation.

The monovalent thallous ion (Tl) was evaluated at the frog end plate in vitro with intracellular microelectrodes. Recordings included end plate potentials (EPPs), and miniature end plate potentials (MEPPs). Replacement of extracellular potassium (K) by 2.5 mM Tl (a) caused increases in MEPP and EPP amplitudes, MEPP frequency, and quantal content, and (b) caused complete recovery of the EPP facilitation index at BAPTA-loaded nerve terminals. Tl's effects were reversible and concentration dependent, and persisted for > 3 h. The increase in MEPP frequency and its rate of decline due to Tl washout were more pronounced at 0 calcium (Ca)-2 mM EGTA than at 0.3 mM EGTA, suggesting that Tl's effects were not due to elevation of internal Ca. Unlike heavy metal ions reportedly capable of substituting for Ca, 0.2 mM Tl did not block, but further enhanced, elevated MEPP frequencies, occurring after nerve stimulation or in high K, to greater levels with barium (Ba) than with Ca. 200 nM omega-conotoxin (omega-CTX) blocked Tl's effect, indicating that Tl primarily entered the nerve terminal via Ca channels. A 50% reduction in sodium (Na) did not modify Tl's effect, although removal of K in the presence of 20 microM ouabain and 2.5 mM Tl caused an exaggerated increase in MEPP frequency, which decreased with a 50% reduction in Na. Based on the analysis, Tl neither substituted for Ca nor elevated internal Ca and Na, nor were its effects antagonized by ouabain; Tl increased quantal secretion, possibly by a fusogenic mechanism, after its entry into the nerve terminal.     

Ni2+ block of CaV3.1 (alpha1G) T-type calcium channels

Ni(2+) inhibits current through calcium channels, in part by blocking the pore, but Ni(2+) may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni(2+) on Ca(V)3.1 is affected by permeant ions. We find two components to block by Ni(2+), a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent K(d) = 3 vs. 1 mM Ni(2+), with 2 mM Ca(2+) or Ba(2+)) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca(2+) or Ba(2+)). Slow block depends both on the concentration and on the identity of the permeant ion (Ca(2+) vs. Ba(2+) vs. Na(+)). Slow block is 2-3x faster in Ba(2+) than in Ca(2+) (2 or 110 mM), and is approximately 10x faster with 2 vs. 110 mM Ca(2+) or Ba(2+). Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations ( approximately 3 muM Ca(2+)). We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore. The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore. In contrast to the complex pore block observed for Ca(V)3.1, inhibition of Ca(V)3.2 by Ni(2+) was essentially independent of voltage, and was similar in 2 mM Ca(2+) vs. Ba(2+), consistent with inhibition by a different mechanism, at a site outside the pore.     

Calcium dependence of uni-quantal release latencies and quantal content at mouse neuromuscular junction

Uni-quantal endplate currents (EPC) were recorded at mouse diaphragm neuromuscular synapse by extracellular microelectrode during motor nerve stimulation. The probability of release expressed as quantal content m(o), and variability of synaptic latencies expressed as P90 were estimated in the presence of extracellular calcium ([Ca2+]o) varying between 0.2 and 0.6 mM in the bathing solution. At 0.2 mM ([Ca2+]o), m(o) was low (0.10) and many of long-latency EPCs were present during the late phase of the release (P90 = 2.44 ms). No change in m(o) was found when ([Ca2+]o) was 0.3 mM, but P90 decreased by 39 %. For latency shortening, saturating concentration of ([Ca2+]o) was 0.4 mM, when P90 was 1.49 ms and latencies did not further change at 0.5 and 0.6 mM ([Ca2+]o). In the latter concentrations, however, an increase of m(o) was still observed. It can be concluded that the early phase of the secretion did not significantly change when ([Ca2+]o) was raised and that only the late phase of the release depends on extracellular calcium up to 0.4 mM.     

Characteristics of anion channels in the tonoplast of the liverwort Conocephalum conicum

Isolated vacuoles of the liverwort Conocephalum conicum thallus cells were investigated using the patch-clamp technique. At high cytosolic Ca(2+) activities, slowly activating currents were evoked by positive potentials. The currents were conducted by the SV (slow-vacuolar) channel. When isolation of vacuoles was carried out at high Mg(2+) and low Ca(2+) concentration and the same proportion of the cations was kept in the bath, currents were recorded at negative potentials. Once activated, these currents persisted even after replacing Mg(2+) with K(+) in the bath. Sr(2+) and Ba(2+) were also effective activators of the currents. With a Cl(-) gradient, 10 mM in the bath and 100 mM in the lumen, currents were significantly reduced and the current-voltage characteristics shifted towards the reversal potential of Cl(-), indicating Cl(-) selectivity. Currents almost vanished after substituting Cl(-) with gluconate. They were strongly reduced by anion channel inhibitors 4,4'-diisothicyanatostilbene-2,2'-disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (A9C; 2 mM) and ethacrinic acid (0.5 mM). Single-channel recordings revealed a 32 pS channel activating at negative voltages. It is concluded that the currents at negative potentials are carried by anion channels suitable for conducting anions from the cytosol to the vacuole. The anion channels were weakly calcium dependent, remaining active at physiological calcium concentration. The channels were almost equally permeable to Cl(-), NO3(-) and SO4(2-), and much less permeable to malate(2-). Anion channels did not respond to ATP addition. cAMP (10 microM) had a weak effect on anion channels. Protein kinase A (0.4 U) added to the medium caused no significant effect on anion channels.     

The role of calcium in modulation of the kinetics of synchronous and asynchronous quantal release at the neuromuscular junction

To elucidate the mechanisms of calcium regulation of the kinetics of the evoked neurotransmitter quantal release, we have investigated the temporal parameters of acetylcholine secretion in the mouse neuro-muscular junction at varying extracellular calcium concentration, in the presence of calcium channel blockers or intracellular calcium buffers. Acetylcholine secretion was induced by the motor nerve stimulation at a low frequency, which did not produce facilitation of the neurotransmitter release. The analysis of histograms of synaptic delays of uniquantal endplate currents recorded during 50 ms after the presynaptic action potential revealed three components of the secretion process: early and late periods of synchronous release and a delayed asynchronous release. At reduced extracellular calcium level, the relative number of quanta released during the asynchronous phase of secretion increased, while the rate of quantal release during the early synchronous period decreased. The findings support the hypothesis of participation of low- and high-affinity calcium sensors with different calcium binding kinetics in regulation of, respectively, synchronous and asynchronous release of neurotransmitter quanta.     

On the effects of divalent cations and ethylene glycol-bis-(beta- aminoethyl ether) N,N,N'',N''-tetraacetate on action potential duration in frog heart

Resting and action potentials were recorded from superfused strips of frog ventricle. Reducing the bathing calcium concentration ([Ca2+]0) with or without ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) prolongs the action potential (AP). The change in the duration of the AP extends over many minutes, but is rapidly reversed by restoring calcium ions. Other changes (e.g., in resting potential and overshoot) are, however, only more slowly reversed. Reducing [Ca2+]0 with 0.2, 2, or 5 mM EGTA produces progressively greater prolongation of AP; maximum values were well in excess of 1 min. This prolongation can be reversed by other divalent cations in EGTA (Mg2+, Sr2+) or Ca-free (Mn2+) solutions, or by acetylcholine. Barium ions increase AP duration in keeping with their known effect on potassium conductance. D600, which blocks the slow inward current in cardiac muscle, is without effect on the action potentials recorded in EGTA solutions, or on the time course and extent of the recovery to normal duration upon restoring calcium ions. It is concluded that divalent cations exert an influence on membrane potassium conductance extracellularly in frog heart. The cell membrane does not become excessively "leaky" in EGTA solutions.     

Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.     

TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) > Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.     

Changes in the Asynchronicity of Transmitter Release at the Neuromuscular Junction during Prolonged High-Frequency Stimulation

In experiments on neuromuscular junctions in the frog m. cutaneous-pectoris, changes in the intensity and asynchronicity of transmitter release during high-frequency (10 and 50 sec^-1) rhythmic stimulation of the motor nerve were investigated using extracellular recording. At low extracellular Ca²⁺ concentrations, rhythmic stimulation resulted in a gradual enlargement of the quantum content of end-plate currents (EPC), the so-called facilitation. The latter phenomenon was accompanied by an increase in the average value and variance of synaptic delays of single-quantum EPC, a shift of the main mode of their distribution towards greater values, and an increase in the latency of the nerve ending responses. The above-described changes reduce the magnitude of facilitation in the neuromuscular synapse.     

Modulation of the gating of unitary cardiac L-type Ca(2+) channels by conditioning voltage and divalent ions

Although a considerable number of studies have characterized inactivation and facilitation of macroscopic L-type Ca(2+) channel currents, the single channel properties underlying these important regulatory processes have only rarely been examined using Ca(2+) ions. We have compared unitary L-type Ca(2+) channel currents recorded with a low concentration of Ca(2+) ions with those recorded with Ba(2+) ions to elucidate the ionic dependence of the mechanisms responsible for the prepulse-dependent modulation of Ca(2+) channel gating kinetics. Conditioning prepulses were applied across a wide range of voltages to examine their effects on the subsequent Ca(2+) channel activity, recorded at a constant test potential. All recordings were made in the absence of any Ca(2+) channel agonists. Moderate-depolarizing prepulses resulted in a decrease in the probability of opening of the Ca(2+) channels during subsequent test voltage steps (inactivation), the extent of which was more dramatic with Ca(2+) ions than Ba(2+) ions. Facilitation, or increase of the average probability of opening with strong predepolarization, was due to long-duration mode 2 openings with Ca(2+) ions and Ba(2+) ions, despite a decrease in Ca(2+) channel availability (inactivation) under these conditions. The degree of both prepulse-induced inactivation and facilitation decreased with increasing Ba(2+) ion concentration. The time constants (and their proportions) describing the distributions of Ca(2+) channel open times (which reflect mode switching) were also prepulse-, and ion-dependent. These results support the hypothesis that both prior depolarization and the nature and concentration of permeant ions modulate the gating properties of cardiac L-type Ca(2+) channels.