Omega Conus geographus toxin: a peptide that blocks calcium channels

We previously reported that omega Conus geographus toxin (omega CgTX), blocks evoked-release of transmitter at synapses in frog and attenuates the Ca2+ component of the action potential of chick dorsal root ganglion neurons. We report here voltage-clamp experiments on cultured chick dorsal root ganglion neurons which demonstrate that omega CgTX produces a persistent block of voltage-gated Ca2+ currents. Thus, we conclude that omega CgTX inhibits synaptic transmission by blocking Ca2+ channels in the presynaptic nerve terminal. The toxin had no effect on K+ currents; however, in some but not all neurons, omega CgTX reduced Na+ currents by 10-25%. These findings suggest that omega CgTX should be useful as a probe to examine synaptic Ca2+ channels.     

Charybdotoxin block of Shaker K+ channels suggests that different types of K+ channels share common structural features

Charybdotoxin (CTX), a 37 amino acid protein isolated from the venom of L. quinquestriatus, is a high-affinity blocker of various Ca2(+)-activated K+ channels. CTX also blocks Drosophila Shaker (Sh) clone H4 transient K+ currents expressed in Xenopus oocytes with similar affinity (Kd = 3.6 nM). CTX blocks both the open and the closed states of Sh channels with no apparent change in gating behavior. In addition, the block is enhanced as the ionic strength is lowered. These properties are identical to those of CTX block of Ca(+)-activated K+ channels, and these results suggest that the external pore openings of these two functionally dissimilar K+ channels may share common structural features.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca(2+) channels are important structural determinants for the passage of Ca(2+) across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a 1S subunit of the skeletal L-type channel (Ca(v)1.1) to lysine virtually eliminates passage of Ca(2+) during step depolarizations. In this study, we examined the ability of this mutant Ca(v)1.1 channel (SkEIIIK) to conduct inward Na(+) current. When 150 mM Na(+) was present as the sole monovalent cation in the bath solution, dysgenic (Ca(v)1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na(+). Ca(2+) block of SkEIIIK-mediated Na(+) current was revealed by the substantial enhancement of Na(+) current amplitude after reduction of Ca(2+) in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na(+) currents through the mutant Ca(v)1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na(+) channel when Na(+) is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca(2+) permeability mediated by Ca(v) channels on physiological processes that extend beyond channel gating and permeability.     

Action of quinidine on ionic currents of molluscan pacemaker neurons

The effects of quinidine on the fast, the delayed, and the Ca2+- activated K+ outward currents, as well as on Na+ and Ca2+ inward currents, were studied at the soma membrane from neurons of the marine mollusk Aplysia californica. External quinidine blocks these current components but to different degrees. Its main effect is on the voltage- dependent, delayed K+ current, and it resembles the block produced by quaternary ammonium ions (Armstrong, C. M., 1975, Membranes, Lipid Bilayers and Biological Membranes: Dynamic Properties, 3:325-358). The apparent dissociation constant is 28 microM at V = +20 mV. The blocking action is voltage and time dependent and increases during maintained depolarization. The data are consistent with the block occurring approximately 70-80% through the membrane electric field. Internal injection of quinidine has an effect similar to that obtained after external application, but its time course of action is faster. External quinidine may therefore have to pass into or through the membrane to reach a blocking site. The Ca2+-activated K+ current is blocked by external quinidine at concentrations 20-50-fold higher compared with the delayed outward K+ current. In addition, it prolongs the time course of decay of the Ca2+-activated K+ current. Na+ and Ca2+ inward currents are also blocked by external quinidine, but again at higher concentrations. The effects of quinidine on membrane currents can be seen from its effect on action potentials and the conversion of repetitive "beating" discharge activity to "bursting" pacemaker activity.     

A store-operated calcium channel in Drosophila S2 cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ > Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.     

Selectivity of omega-CgTx-MVIIC toxin from Conus magus on calcium currents in enteric neurons.

Whole-cell perforated patch clamp recordings were used to analyze selectivity of omega-CgTx-MVIIC toxin for voltage-dependent calcium currents in cultured myenteric neurons from guinea-pig small intestine. Omega-CgTx-MVIIC (300 nM) blocked 37 +/- 9% of the peak current activated from -80 mV in 15 neurons by mostly affecting the plateau phase of the current. The toxin suppressed peak current activated from -30 mV dose-dependently with an IC50 of 70 +/- 8 nM. The blockade was complete at toxin concentrations of 1 microM. Thus, it appears that omega-CgTx-MVIIC blocks high voltage activated (HVA) calcium channels in the myenteric neurons unselectively as well as other types of HVA Ca2+ channels including P and Q channels.     

Omega-conotoxin GVIA blocks a Ca2+ current in bovine chromaffin cells that is not of the "classic" N type.

Previous studies have identified two components of whole-cell Ca2+ current in bovine chromaffin cells. The "standard" component was activated by single depolarizations, while "facilitation" could be activated by large prepulses or repetitive depolarizations. Neither current component was sensitive to changes in holding potential between -100 and -50 mV; thus neither appeared to be carried by N-type Ca2+ channels. We now report that the facilitation Ca2+ current is insensitive to omega-conotoxin GVIA (omega-CgTx), but that the toxin blocks approximately 50% of the standard Ca2+ current. In some cells the toxin blocks all of the standard Ca2+ current, in others about half of the current, while in others it has no effect. Kinetic differences in current activation are observed after toxin application. These results suggest that the standard component of chromaffin cell Ca2+ current is composed of two pharmacologically distinct channels-one is omega-CgTx sensitive and the other is not. Two kinetically distinct types of 14 pS Ca2+ channels that may correspond to the omega-CgTx-sensitive and -insensitive components were observed in single-channel experiments. Because omega-CgTx blocked Ca2+ channels that were not inactivated by a depolarized holding potential, the commonly used Ca2+ channel categorization scheme may be inadequate to describe the Ca2+ channels found in chromaffin cells.     

Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons

The action of charybdotoxin (ChTX), a peptide component isolated from the venom of the scorpion Leiurus quinquestriatus, was investigated on membrane currents of identified neurons from the marine mollusk, Aplysia californica. Macroscopic current recordings showed that the external application of ChTX blocks the Ca-activated K current in a dose- and voltage-dependent manner. The apparent dissociation constant is 30 nM at V = -30 mV and increases e-fold for a +50- to +70-mV change in membrane potential, which indicates that the toxin molecule is sensitive to approximately 35% of the transmembrane electric field. The toxin is bound to the receptor with a 1:1 stoichiometry and its effect is reversible after washout. The toxin also suppresses the membrane leakage conductance and a resting K conductance activated by internal Ca ions. The toxin has no significant effect on the inward Na or Ca currents, the transient K current, or the delayed rectifier K current. Records from Ca-activated K channels revealed a single channel conductance of 35 +/- 5 pS at V = 0 mV in asymmetrical K solution. The channel open probability increased with the internal Ca concentration and with membrane voltage. The K channels were blocked by submillimolar concentrations of tetraethylammonium ions and by nanomolar concentrations of ChTX, but were not blocked by 4-aminopyridine if applied externally on outside-out patches. From the effects of ChTX on K current and on bursting pacemaker activity, it is concluded that the termination of bursts is in part controlled by a Ca-activated K conductance.     

Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells

Single channel currents through Ca2+-activated K+ channels of bovine chromaffin cells were measured to determine the effects of small ions on permeation through the channel. The channel selects strongly for K+ over Na+ and Cs+, and Rb+ carries a smaller current through the channel than K+. Tetraethylammonium ion (TEA+) blocks channel currents when applied to either side of the membrane; it is effective at lower concentrations when applied externally. Millimolar concentrations of internal Na+ reduce the average current through the channel and produce large fluctuations (flicker) in the open channel currents. This flickery block is analyzed by a new method, amplitude distribution analysis, which can measure block and unblock rates in the microsecond time range even though individual blocking events are not time-resolved by the recording system. The analysis shows that the rate of block by Na+ is very voltage dependent, but the unblock rate is voltage independent. These results can be explained easily by supposing that current flow through the channel is diffusion limited, a hypothesis consistent with the large magnitude of the single channel current.     

Different types of K+ channel current are generated by different levels of a single mRNA.

A cloned human voltage-sensitive K+ channel HLK3 which is present in T-lymphocytes and in the brain was expressed in Xenopus oocytes and after permanent transfection of a human B-lymphocyte cell line (IM9). Injections of low cRNA concentrations into Xenopus oocytes led to the expression of a transient K+ current, with saturating current-voltage (I-V) relationship, which was abolished by repetitive stimulations due to a slow recovery from inactivation. This transient K+ channel current was fully inhibited by 10 nM charybdotoxin. Injection of high concentrations of the same RNA led to a non-inactivating K+ current, with linear I-V curve, which did not undergo use-dependent inactivation and was hardly sensitive to 10 nM charybdotoxin. Intermediate behaviour due to changing proportions of these two types of K+ channel expression were observed at intermediate RNA concentrations. Transient and non-inactivating K+ currents were also observed by both whole-cell and single channel patch-clamp recording from HLK3 transfected IM9 cells. The main conductance of the channel in the two different modes (inactivating and charybdotoxin-sensitive or non-inactivating and charybdotoxin-resistant) is the same (12-14 pS). Destruction of the cytoskeletal elements with cytochalasin D, colchicine or botulinum C2 toxin in oocyte experiments prevented expression of the sustained mode of the K+ channel. The results suggest that the sustained mode obtained at high RNA concentrations corresponds to channel clustering involving cytoskeletal elements. This differential functional expression of K+ channels associated with different levels of mRNA appears as a new important factor to explain the biophysical and pharmacological diversity of voltage-sensitive K+ channels.     

Sustained and transient calcium currents in horizontal cells of the white bass retina

Calcium currents were recorded from cultured horizontal cells (HCs) isolated from adult white bass retinas, using the whole-cell patch-clamp technique. Ca2+ currents were enhanced using 10 mM extracellular Ca2+, while Na+ and K+ currents were pharmacologically suppressed. Two components of the Ca2+ current, one transient, the other sustained, were found. The large transient component of the Ca2+ current, which has not been seen before in HCs, is similar, but not identical, to the T-type Ca2+ current described previously in a variety of preparations. The sustained component of the Ca2+ current is similar, but not identical, to the L-type current described in other preparations. FTX, a factor isolated from the venom of the funnel-web spider, Agelenopsis aperta, preferentially and irreversibly blocks the sustained component of the Ca2+ current at very dilute concentrations. The sustained component of the Ca2+ current inactivates slowly, over the course of 15-60 s, in some HCs. This inactivation of the sustained Ca2+ current, when present, is primarily voltage dependent rather than Ca2+ dependent.     

Action potential waveforms reveal simultaneous changes in ICa and IK produced by 5-HT in rat dorsal raphe neurons.

Action potentials were recorded from serotonergic dorsal raphe (DR) neurons acutely isolated from the adult rat brain. Action potential waveforms were used as command potentials for whole-cell patch-clamp studies to investigate the Ca2+ and K+ currents underlying action potentials and the modulatory effects of 5-Hydroxytryptamine (5-HT) on them. These data were compared with currents elicited by using rectangular voltage steps of the type commonly used in voltage-clamp experiments. In the same cell, 5-HT simultaneously augmented K+ currents and inhibited Ca2+ currents. Experimental conditions were chosen which allowed us to examine the action of 5-HT on K+ and Ca2+ currents simultaneously or in isolation; 5-HT produced a larger inhibition of calcium current during an action potential waveform compared with that measured by using rectangular steps of voltage. A possible explanation for this finding is that the maximal inhibition is seen immediately after a voltage jump and then decreases with time. Action potentials are, in general, so brief that little time-dependent relief of block is observed. Most of the inhibition of Ca2+ current resulted from a direct effect on Ca2+ channels rather than a shortening of the action potential. The inhibition of Ca2+ current by 5-HT also decreased the Ca(2+)-activated K+ currents. These results suggest that 5-HT reduces DR neuron excitability by the simultaneous activation of K+ channel currents open at the resting potential and the suppression of Ca2+ channel currents.     

Conversion of beating to bursting pacemaker activity: Action of quinidine

External quinidine converts the pacemaker neurone L-11, found in the Aplysia abdominal ganglion, from spontaneously "beating" to "bursting" discharge activity. Quinidine-induced bursting ceased when entry of Ca2+ ions into the cells was blocked in a Ca2+-free, Co2+-containing solution or if internal Ca2+ accumulation was prevented by the injection of EGTA. The analysis of membrane currents from voltage clamp experiments showed that quinidine blocks the Ca2+ inward current in a dose- and time-dependent manner. In addition, the currents were displaced to the left on the voltage axis, causing an increase of the inward current at negative membrane potentials. External quinidine suppresses the Ca2+-activated K+ current induced by intracellular Ca2+ injections and acts to prolong its decay phase. The slowing of the decay phase of the Ca2+-activated K+ current by quinidine was prevented after intracellular injection of EGTA, indicating that Ca2+ removal is impaired by the drug. It is suggested that the increase of Ca2+ inward current at negative potentials and the prolonged activation of the Ca2+-activated K+ current play a major role in causing the bursting discharge behavior in normally beating cells.     

Block of kainate receptor channels by Ca2+ in isolated spinal trigeminal neurons of rat.

Compared with N-methyl-D-aspartate-activated channels, the interaction of Ca2+ with kainate-activated or with quisqualate-activated channels is not well understood. We have studied the effect of Ca2+ on kainate-activated currents in isolated trigeminal neurons and found that Ca2+ inhibits kainate responses. This inhibition occurs not because Ca2+ changes the affinity of kainate to its receptor, but because Ca2+ blocks monovalent cation permeation through kainate-activated channels. This Ca2+ block gives rise to the outward rectification of the kainate responses.     

Depolarization-induced slowing of Ca2+ channel deactivation in squid neurons.

Properties of squid giant fiber lobe (GFL) Ca2+ channel deactivation (closing) were studied using whole-cell voltage clamp. Tail currents displayed biexponential decay, and fast and slow components of these tails exhibited similar external Ca(2+)- and voltage-dependence. Both components also shared similar inactivation properties. Increasing duration pulses to strongly depolarizing potentials caused a substantial slowing of the rate of deactivation for the fast component, and also led to an apparent conversion of fast tail currents to slow without an increase in total tail amplitude. A five-state kinetic model that computed the closing of channels differentially populating two open states could simulate the kinetic characteristics of GFL Ca2+ pulse and tail currents over a wide voltage range. The kinetics of the proposed state transition was very similar to the time course of relief of omega-Agatoxin IVA Ca2+ channel block with long pulses. A similar model predicted that the relief of block could occur via faster toxin dissociation from the second open state. Thus, GFL Ca2+ channels possess a unique form of voltage-dependent gating modification, in which maintained prior depolarization leads to a significant delay to channel closure at negative potentials. At the nerve terminal, amplified Ca2+ signals generated by such a mechanism might alter synaptic responses to repetitive stimulation.     

Voltage gated ionic channels in rat cultured astrocytes, reactive astrocytes and an astrocyte-oligodendrocyte progenitor cell

Astrocytes (both type 1 and type 2), cultured from the central nervous system of newborn or 7 day old rats show voltage gated sodium and potassium channels that are activated when the membrane is depolarized to greater than -40 mV. The sodium channels in these cells have an h-infinity curve similar to that of nodal membranes but the activation (peak current-voltage) curves are shifted along the voltage axis by about +30 mV. These sodium currents are blocked only by high concentrations of tetrodotoxin. The voltage activated potassium currents in both types of astrocyte show at least two components; an inactivating component that is suppressed at holding potentials of greater than -40 mV and a persistent, non-inactivating current. Several types of single channel currents were observed in outside-out membrane patches from type 2 astrocytes. One type of potassium channel showed inactivation on depolarization and may contribute to the whole-cell inactivating current. In contrast, oligodendrocytes showed no obvious voltage gated membrane channels. The properties of the type 2 astrocyte-oligodendrocyte progenitor cell were investigated in two ways: 1) by examination of cells just beginning to differentiate along the "electrically silent" oligodendrocyte pathway or 2) by recording from progenitor cells cultured for 24 hours in the presence of cycloheximide to block the appearance of new membrane channels. In both cases, voltage gated inward (sodium) and outward (potassium) currents were noted. The outward current response showed both an inactivating and a non-inactivating component. Similar voltage activated inward and outward membrane currents were noted in reactive astrocytes freshly isolated (3-6 hours) from lesioned areas of adult rat brains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons

The major component of whole-cell Ca2+ current in differentiated, neuron-like rat pheochromocytoma (PC12) cells and sympathetic neurons is carried by dihydropyridine-insensitive, high-threshold-activated N-type Ca2+ channels. We show that these channels have unitary properties distinct from those of previously described Ca2+ channels and contribute both slowly inactivating and large sustained components of whole-cell current. The N-type Ca2+ currents are modulated by GTP binding proteins. The snail toxin omega-conotoxin reveals two pharmacological components of N-type currents, one blocked irreversibly and one inhibited reversibly. Contrary to previous reports, neuronal L-type channels are insensitive to omega-conotoxin. N-type Ca2+ channels appear to be specific for neuronal cells, since their functional expression is greatly enhanced by nerve growth factor.     

Characterization and localization of two ion-binding sites within the pore of cardiac L-type calcium channels

L-type Ca channels from porcine cardiac sarcolemma were incorporated into planar lipid bilayers. We characterized interactions of permeant and blocking ions with the channel's pore by (a) studying the current-voltage relationships for Ca2+ and Na+ when equal concentrations of the ions were present in both internal and external solutions, (b) testing the dose-dependent block of Ba2+ currents through the channels by internally applied cadmium, and (c) examining the dose and voltage dependence of the block of Na+ currents through the channels by internally and externally applied Ca2+. We found that the I-V relationship for Na+ appears symmetrical through the origin when equal concentrations of Na+ are present on both sides of the channel (gamma = 90 pS in 200 mM NaCl). The conductance for outward Ca2+ currents with 100 mM Ca2+ on both sides of the channel is approximately 8 pS, a value identical to that observed for inward currents when 100 mM Ca2+ was present outside only. This provides evidence that ions pass through the channel equally well regardless of the direction of net flux. In addition, we find that internal Cd2+ is as effective as external Cd2+ in blocking Ba2+ currents through the channels, again suggesting identical interactions of ions with each end of the pore. Finally, we find that micromolar Ca2+, either in the internal or in the external solution, blocks Na+ currents through the channels. The affinity for internally applied Ca2+ appears the same as that for externally applied Ca2+. The voltage dependence of the Ca(2+)-block suggests that the sites to which Ca2+ binds are located approximately 15% and approximately 85% of the electric field into the pore. Taken together, these data provide direct experimental evidence for the existence of at least two ion binding sites with high affinity for Ca2+, and support the idea that the sites are symmetrically located within the electric field across L-type Ca channels.     

Transmitter release in botulinum-poisoned muscles

Examination of miniature end-plate potentials (m.e.p.ps) in rat skeletal muscle poisoned in vivo by botulinum toxin type A reveals the presence of two populations of potentials. One population which corresponds to m.e.p.ps in unpoisoned muscles and to quantal end-plate potentials. The frequency of these m.e.p.ps is greatly reduced by botulinum toxin. The second population of m.e.p.ps has quite different characteristics. These m.e.p.ps have a more variable, but generally much larger amplitude, and their time to peak is longer than normal m.e.p.ps. The frequency of these m.e.p.ps increases during poisoning and reaches 0.3-1 Hz after 10-14 days. In addition to the variability in amplitude and time-to-peak these m.e.p.ps differ from those at unpoisoned junctions by being unaffected by procedures which alter extra- or intracellular Ca2+ concentrations. The appearance of this Ca2+-insensitive spontaneous quantal secretion of acetylcholine is apparently not a direct effect of the toxin but secondary to blockade of impulse transmission since it also appears at unpoisoned end-plates when transmission is impaired for other reasons. Procedures which increase the intracellular Ca2+ concentration in nerve terminals restore transmitter release from botulinum toxin poisoned nerves. Furthermore, the block caused by the toxin is very temperature-dependent, a reduction in temperature relieving the block. Since presynaptic Ca2+ currents are unaltered by the toxin it is proposed that the block of transmission is due to a reduction in the calcium content of the nerve terminal to a level where the amount of Ca2+, which normally enters, is insufficient to activate transmitter release.     

A Fast Transient Outward Monovalent Current in Rat Saphenous Myocytes Passing Through Ca2+ Channels

Transient outward currents in rat saphenous arterial myocytes were studied using the perforated configuration of the patch-clamp method. When myocytes were bathed in a Na-gluconate solution containing TEA to block large-conductance Ca2+-activated K+ (BK) currents, depolarizing pulses positive to +20 mV from a holding potential of -100 mV induced fast transient outward currents. The activation and inactivation time constants of the current were voltage dependent, and at +40 mV were 3.6 +/- 0.8 ms and 23.9 +/- 6.4 ms (n = 4), respectively. The steady-state inactivation of the transient outward current was steeply voltage dependent (z = 1.7), with 50% of the current inactivated at -55 mV. The current was insensitive to the A-type K+ channel blocker 4-AP (1-5 mM), and was modulated by external Ca, decreasing to approximately 0.85 of control values upon raising Ca2+ from 1 to 10 mM, and increasing approximately 3-fold upon lowering it to 0.1 mM. Transient outward currents were also recorded following replacement of internal K+ with either Na+ or Cs+, raising the possibility that the current was carried by monovalent ions passing through voltage-gated Ca2+ channels. This hypothesis was supported by the finding that the transient outward current had the same inactivation rate as the inward Ba2+ current, and that both currents were effectively blocked by the L-type Ca2+ channel blocker, nifedipine and enhanced by the agonist BAYK8644.