Phytochrome activation of k channels and chloroplast rotation in mougeotia: action spectra.

The action spectra for K(+) channel activation and chloroplast rotation are shown to be similar. Both phenomena exhibit activation at 660 nanometers, inhibition at 740 nanometers, and partial activation at 460 to 500 nanometers. This confirms that K(+) channels in Mougeotia are regulated by phytochrome, and indicates that both phenomena share at least part of the same transduction pathway.

Fractal filtering of channel data.

The fractal dimension of subsets of time series data can be used to modulate the extent of filtering to which the data is subjected. In general, such fractal filtering makes it possible to retain large transient shifts in baseline with very little decrease in amplitude, while the baseline noise itself is markedly reduced (Strahle, W.C. (1988) Electron. Lett. 24, 1248-1249). The fractal filter concept is readily applicable to single channel data in which there are numerous opening/closing events and flickering. Using a simple recursive filter of the form: Yn = w.Yn-1 + (1 - w)Xn, where Xn is the data, Yn the filtered result, and w is a weighting factor, 0 less than w less than 1, we adjusted w as a function of the fractal dimension (D) for data subsets. Linear and ogive functions of D were used to modify w. Of these, the ogive function: w = [1 + p(1.5-D)]-1 (where p affects the amount of filtering), is most useful for removing extraneous noise while retaining opening/closing events.

Calcium activation of mougeotia potassium channels.

Phytochrome mediates chloroplast movement in the alga Mougeotia, possibly via changes in cytosolic calcium. It is known to regulate a calcium-activated potassium channel in the algal plasma membrane. As part of a characterization of the potassium channel, we examined the properties of calcium activation. The calcium ionophore A23187 activates the channel at external [Ca(2+)] as low as 20 micromolar. However, external [Ca(2+)] is not required for activation of the channel by photoactivated phytochrome. Furthermore, when an inhibitor of calcium release from internal stores, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), is present, red light no longer stimulates channel activity. We conclude that phytochrome activates the plasma membrane potassium channel by releasing calcium from intracellular calcium vesicles; the elevated cytosolic calcium then stimulates channel activity by an unknown mechanism. In the presence of TMB-8, red light does induce chloroplast rotation; thus, potassium channel activation may not be coupled to chloroplast rotation.

Red light regulates calcium-activated potassium channels in mougeotia plasma membrane.

The alga Mougeotia has a large central chloroplast whose positioning is regulated by photoactivation of phytochrome, possibly via modulation of cytosolic calcium (Serlin B, Roux SJ [1984] Proc Natl Acad Sci USA 81: 6368-6372). We used the patch clamp technique to examine the effects of red and far-red light on ion channel activity in the plasma membrane of Mougeotia protoplasts to determine if ion channels play a role in chloroplast movement. Patch clamping in the cell-attached mode reveals two channels of about 2 and 4 picoamperes amplitude at 0 millivolt (inside pipette) and estimated conductances of 30 and 65 picosiemens. They are activated by red light irradiation after a lag period of about 2 to 5 minutes. Far-red light, when applied immediately after red light irradiation, reverses this activation; otherwise it has no effect. This result implicates phytochrome. The addition of the calcium ionophore, A23187, also activates ion channel activity after a lag of a few minutes. The channels are not specific for calcium since they are present when calcium is removed from the external and pipette media. They are inhibited by quaternary ammonium ions. Thus, we believe they are calcium-activated potassium channels. Their possible role in chloroplast positioning is discussed.

Tetrahydroaminoacridine (THA) as a pharmacological probe in Alzheimer's disease (AD) and other neurodegenerative disorders.

Unlike other potent enhancers of cholinergic function in the central nervous system (CNS), THA appears to sustain improved function in many moderately impaired AD patients when the Summers procedure is followed. THA has a complex pharmacology. In addition to its enhancement of cholinergic transmission a hydroxylated metabolite might chelate aluminum (A1), thereby removing multiple toxicological constraints on CNS function. This mobilized THA metabolite-A1 complex might either be re-distributed to less sensitive sites or removed from the CNS across the blood-brain barrier (BBB). Since the known presence of A1 in AD brain is not necessarily causal, a positivistic approach to research and treatment with THA and its metabolites might serve to clarify this difficult and challenging problem.

Tetrahydroaminoacridine blocks potassium channels and inhibits sodium inactivation in Myxicola.

In voltage-clamped Myxicola giant axons internally and externally applied tetrahydroaminoacridine (THA) blocked K+ channels with a dissociation constant of 100 microM and slowed their rate of activation. At a concentration of 10 microM, internal THA primarily slowed inactivation of conducting Na+ channels. At 100 microM the decline of the Na+ current during depolarizing pulses was biphasic, with an initial phase 2 to 3 times faster than in control axons. In the presence of THA there was a steady-state inward current accompanied by an increase in amplitude and time constant of Na+ tail currents, as if THA blocked Na+ channels by first entering them and then rendered THA-occluded channels resistant to fast inactivation. THA did not alter activation, prepulse-induced fast inactivation or slow inactivation. The effects of THA on voltage-dependent axonal ion channels might account for central nervous system hyperexcitability seen in some patients treated with THA. Because THA is a potent, centrally active anticholinesterase, even subtle ion channel-directed effects might contribute to its putative antidementia action in clinical states involving a central nervous system deficiency of acetylcholine by selective augmentation of acetylcholine release and/or negation of autoreceptor effects of endogeneous acetylcholine.

Properties of Single K and Cl Channels in Asclepias tuberosa Protoplasts.

Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K(+) and Cl(-), respectively. Whole cell K(+) currents activated exponentially during step depolarizations, while voltage-dependent Cl(-) channels were activated by hyperpolarizations. Single K(+) channel conductance was 40 +/- 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs(+) and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 +/- 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn(2+) and ethacrynic acid. Whole-cell Cl(-) currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development.

Zonisamide enhances slow sodium inactivation in Myxicola.

In voltage-clamped Myxicola giant axons Zonisamide (1,2-benzisoxazole-3-methanesulfonamide) caused a hyperpolarizing shift in the steady-state fast inactivation curve and retarded recovery from fast and slow Na+ inactivation. The effects of Zonisamide on steady-state fast inactivation could be described assuming a single binding site with a dissociation constant of 12 microM. Slow inactivation was significantly more sensitive, with a Kd of 1 microM from both steady-state and kinetic data. While these results account for anticonvulsant activity, the differential sensitivity suggests Zonisamide may also be useful in studies of the slow inactive state of the Na+ channel.

Anticonvulsants modify inactivation but not activation processes of sodium channels in Myxicola axons.

The effects of pronase and the anticonvulsant drugs diphenylhydantoin, bepridil, and sodium valproate on fast and slow Na+ inactivation were examined in cut-open Myxicola giant axons with loose patch-clamp electrodes applied to the internal surface. Pronase completely eliminated fast Na+ inactivation without affecting the kinetics of Na+ activation or the maximum Na+ conductance. The time and voltage dependences of slow inactivation following pronase treatment were identical to those measured before enzyme application in the same axons. All three anticonvulsants slowed the time course of recovery from fast Na+ inactivation in untreated axons, and shifted the steady-state fast inactivation curve in the hyperpolarizing direction along the voltage axis. Anticonvulsants enhanced steady-state slow inactivation and retarded recovery from slow inactivation in both untreated and pronase-treated axons. Although some quantitative differences were seen, the order of potency of the anticonvulsants on slow Na+ inactivation was the same as that for recovery from fast inactivation.

Dendrotoxin blocks potassium channels and slows sodium inactivation in Myxicola giant axons.

Dendrotoxin (DTX) is known to partially block delayed rectifier K+ channels and enhance neurotransmitter release, but no effects on Na+ channels have been reported. In voltage-clamped Myxicola axons DTX affected both the K+ and Na+ conductances. DTX blocked completely Myxicola K+ channels with a KD of 150 nM and induced slow K+ inactivation. DTX doubled the time constants for inactivation of conducting Na+ channels and gating charge immobilization without altering Na+ activation or the voltage- and time-dependent fast and slow Na+ inactivation induced by depolarizing prepulses. A selective effect on open Na+ channel inactivation provides additional evidence for kinetic models in which resting Na+ channels need not open before being inactivated.

Bepridil and valproate retard Na+ reactivation in Myxicola.

Two drugs were examined, each causing a similar specific modification of Na+ channel inactivation gating when internally applied to voltage-clamped Myxicola giant axons. Bepridil is an antianginal-antiarrhythmic agent with vasodilator and direct cardiac inotropic effects. Sodium valproate has anticonvulsant activity and causes use-dependent inhibition of repetitive firing in CNS neurons. Bepridil and sodium valproate caused a dose-dependent decrease in maximum Na+ conductance (KD = 25 microM for bepridil; KD = 0.5 mM for valproate). More importantly, at half-maximal blocking concentrations both drugs shifted steady state Na+ inactivation in the hyperpolarizing direction (by 30 mV for bepridil; 15 mV for valproate) and slowed the recovery of Na+ channels from inactivation (by 300% for bepridil; 60% for valproate). There was no effect on the K+ conductance, voltage-dependence of Na+ activation, or the time-dependence of inactivation of conducting channels. Neither produced non-inactivating Na+ current during long depolarizing steps.

Effects of abscisic acid on K+ channels in Vicia faba guard cell protoplasts.

Potassium channels were resolved in Vicia faba guard cell protoplasts by patch voltage-clamp. Whole-cell currents and single K+ channels had linear instantaneous current-voltage relations, reversing at the calculated Nernst potential for K+. Whole cell K+ currents activated exponentially during step depolarizations, with half-activation times of 400-450 msec at +80 mV and 90-110 msec at +150 mV. Single K+ channel conductance was 65 +/- 5 pS with a mean open time of 1.25 +/- 0.30 msec at 150 mV. Potassium channels were blocked by internal Cs+ and by external TEA+, but they were insensitive to external 4-aminopyridine. Application of 10 microM abscisic acid increased mean open time and caused long-lasting bursts of channel openings. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology.

Amantadine restores impulse conduction across demyelinated nerve segments.

1. Drugs can be identified that relieve clinical symptoms of demyelinating diseases such as multiple sclerosis based on their ability to alter voltage-dependent ion channels in membranes and restore conduction in demyelinated nerve. Beneficial drugs either slow inactivation of membrane Na+ channels or block K+ channels. 2. Amantadine, an antiviral drug that slows Na+ inactivation in Myxicola giant axons, restores conduction in frog and rat sciatic nerves partially demyelinated by disruption of the perineurium (the 'perineurial window') loose ligatures, or lysolecithin. 3. A positive effect of amantadine on several different animal models supports the need for clinically oriented studies of amantadine and related inactivation-blocking agents in diseases such as multiple sclerosis.

Selective modification of sodium channel gating by solvents and drugs.

In Myxicola heavy water (D2O) does not alter Na+ gating currents, but slows activation and inactivation. In this study, the solvent formamide (5-20% v/v) is shown to proportionately and reversibly block Na+ currents and charge movement, suggesting it may be useful for fractionating gating currents. Formamide- and prepulse-sensitive (inactivating) gating currents were identical, comprising 60-80% of total charge. Both had rising phases and decayed as single exponential functions. Formamide-insensitive and non-inactivating charge movements had no rising phases and decayed slowly with more complex kinetics. Another solvent, dimethylsulfoxide (1% v/v), had no effect on Na+ activation or charge movement, though it did affect inactivation. Amantadine (0.1 mM) did not change Na+ activation or charge movement, but slowed inactivation and shifted the foot of the steady state Na+ inactivation curve. Sotalol (0.1 mM) slowed inactivation, but also inhibited Na+ activation and gating current.

Properties of single Na+ channels in cut-open Myxicola giant axons.

Time- and voltage-dependent behavior of the Na+ conductance in dialyzed intact Myxicola axons was compared with cut-open axons subjected to loose-patch clamp of the interior and to axons where Gigaseals were formed after brief enzyme digestion. Voltage and time dependence of activation, inactivation, and reactivation were identical in whole-axons and loose-patch preparations. Single channels observed in patch-clamp axons had a conductance of 18.3 +/- 2.3 pS and a mean open time of 0.84 +/- 0.12 ms. The time-dependence of Na+ currents found by averaging patch-clamp records was similar to intact axons, as was the voltage dependence of activation. Steady-state inactivation in patch-clamped axons was shifted by an average of 15 mV from that seen in loose-patch or intact axons. Substitution of D2O for H2O decreased single channel conductance by 24 +/- 6% in patch-clamped axons compared with 28 +/- 4% in intact axons, slowed inactivation by 58 +/- 8% compared with 49 +/- 6%, and increased mean open time by 52 +/- 7%. The results confirm observations on macroscopic channel behavior in Myxicola and resemble that seen in other excitable tissues.

Differential sensitivity of amphibian nodal and paranodal K+ channels to 4-aminopyridine and TEA.

Voltage-dependent K+ channels are blocked by several drugs, including 4-aminopyridine (4-AP) and tetraethylammonium (TEA). 4-AP is most widely used to localize K+ channels in mammalian and non-mammalian nerve fibers, but 4-AP and TEA alter various K+ channels and/or preparations in specific ways. The reason is not known, in part because dissociation constants for 4-AP and TEA have not been measured for nodal and internodal K+ channels in the same fibers. Smith and Schauf showed that the density of nodal versus paranodal K+ channels in frog nerves depends on fiber diameter. The size dependence was used to determine the relative sensitivity of nodal and internodal K+ channels to 4-AP and TEA, and to compare voltage- and time-dependent activation. The results show nodal and internodal K+ channels activate similarly. However, internodal channels are selectivity blocked by 4-AP while TEA is more effective on nodal channels. A high sensitivity of internodal K+ channels may explain why 4-AP improves symptoms in diseases such as multiple sclerosis.

Membrane-directed effects of the plant hormones abscisic acid, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid.

This study examines two ways plant hormones might influence membrane processes, effects on overall permeability and modifications of specific ion channels. Abscisic acid (ABA) and indole-3-acetic acid (IAA) greatly enhanced erythritol permeability in mixed egg lecithin bilayers. In single component dioleoylphosphatidylcholine bilayers ABA was less effective than IAA, while 2,4-dichlorophenoxyacetate (2,4-D) did not affect either system or alter their ABA response. In Myxicola axons ABA and IAA had no effect, while 2,4-D (10 uM) caused a depolarizing shift of voltage-dependent Na+ and K+ activation by 25 +/- 4 mV and 15 +/- 3 mV, consistent with internal negative surface charge changes of -0.002 e-/A2 and -0.0007 e-/A2. We conclude that both generalized and ion channel-directed effects may link plant hormones and intracellular regulation.

Selective blockade of components of potassium activation in Myxicola axons.

The K+ conductance in Myxicola giant axons activates in two phases which are pharmacologically separable. The fast phase of K+ activation is specifically inhibited by 4-aminopyridine and by the substitution of D2O for H2O. We suggest Myxicola giant axons, like the amphibian node of Ranvier, may possess more than one variety of K+ channel.

4-Aminopyridine improves clinical signs in multiple sclerosis.

Twelve temperature-sensitive male patients with multiple sclerosis and 5 normal men were monitored before, during, and after the intravenous injection of 7 to 35 mg of 4-aminopyridine (4-AP) in 1- to 5-mg doses, every 10 to 60 minutes. Static quantitative perimetry, flicker-fusion frequency, visual acuity, and videotaped neurological examinations were performed. Ten of the 12 patients showed mild to marked improvement. Vision improved in 7 patients, oculomotor function in 5, and motor function (power, coordination, gait) in 5. Improvements developed gradually within minutes of drug injection at doses as low as 2 mg, and gradually reversed around 2 to 4 hours after the peak drug effect. No effects were observed in 5 patients given saline injections. No serious side effects occurred in either the normal subjects or the patients receiving 4-AP. It is concluded that 4-AP lessens multiple neurological deficits in multiple sclerosis and, furthermore, that the K+ channel is functional in demyelinated central nervous system axons in humans. The improvements with 4-AP are substantial enough to be of transient therapeutic benefit in selected patients.