Block of a Ca(2+)-activated potassium channel by cocaine.

The primary target for cocaine is believed to be monoamine transporters because of cocaine's high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K(+) channel in hippocampal neurons grown in culture (IC(50)=approximately 30 microM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca(2+)-activated K(+ )channels are 261+/-37 microM: (-1)s(-1) and 11451+/-1467 s(-1). The equilibrium dissociation constant (K(B)) for cocaine, determined from single-channel parameters, is 43 microM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca(2+)-dependent K(+) channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.

Electrophysiological analysis of NMDA receptor subunit changes in the aging mouse cortex.

NMDA receptors play an important role in memory processes and plasticity in the brain. We have previously demonstrated a significant decrease in NMDARepsilon2 subunit mRNA and protein with increasing age in the C57Bl/6 mouse frontal cortex. In the present study, two-electrode voltage clamp electrophysiology on Xenopus oocytes injected with total RNA harvested from the frontal cortex of young and old C57Bl mice was used to detect changes in receptor composition during aging. Ifenprodil concentration-response curves, magnesium current-voltage curves, and single channel conductances were determined for native receptors. In addition, ifenprodil and magnesium curves were generated for recombinant NMDA receptors of varying subunit ratios. Ifenprodil dose-response curves for all receptors were biphasic. The low affinity component of the curve increased slightly with age, while the high affinity population decreased, mimicking recombinant receptors with decreasing levels of epsilon2. A decrease in maximal current was also observed in aged animals with decreased levels of epsilon2, although single channel conductances were identical between young and old mice. In addition, an increase in sensitivity to magnesium was observed for receptors from older animals. Results are consistent with the interpretation that the epsilon2 subunit is reduced in older mouse frontal cortex. A change in NMDA receptor subunit composition could influence memory processes during aging.

Induction of vanilloid receptor channel activity by protein kinase C.

Capsaicin or vanilloid receptors (VRs) participate in the sensation of thermal and inflammatory pain. The cloned (VR1) and native VRs are non-selective cation channels directly activated by harmful heat, extracellular protons and vanilloid compounds. However, considerable attention has been focused on identifying other signalling pathways in VR activation; it is known that VR1 is also expressed in non-sensory tissue and may mediate inflammatory rather than acute thermal pain. Here we show that activation of protein kinase C (PKC) induces VR1 channel activity at room temperature in the absence of any other agonist. We also observed this effect in native VRs from sensory neurons, and phorbol esters induced a vanilloid-sensitive Ca2+ rise in these cells. Moreover, the pro-inflammatory peptide, bradykinin, and the putative endogenous ligand, anandamide, respectively induced and enhanced VR activity, in a PKC-dependent manner. These results suggest that PKC may link a range of stimuli to the activation of VRs.

Selective potentiation of L-type calcium channel currents by cocaine in cardiac myocytes.

Cocaine use poses a major health problem not only because of the dependence it causes but also because of the generation of life-threatening cardiac arrhythmias following overdose. Elucidating the molecular mechanisms of action of cocaine, therefore, remains a critical step in developing treatment for cocaine addiction and preventing cardiac complications. Although the neurotransmitter transporters are suggested to be primary targets for cocaine, the continued drug-seeking behavior of transporter knock-out mice suggests the involvement of additional mechanisms. Several studies have shown that voltage-gated calcium channel blockers can prevent the behavioral and reinforcing effects of the drug and also cocaine-induced cardiac events, including lethal ventricular fibrillation. However, the role of voltage-gated calcium channels in cocaine-induced responses is not clear. Herein, I show that cocaine, in pharmacological doses, selectively and potently enhances L-type calcium channel currents in isolated rat ventricular myocytes. This potentiation by cocaine is due to an increase and decrease, respectively, in the calcium channel opening and closing rates, with no apparent effects on voltage-dependence or single-channel conductance. The effects of cocaine are rapidly reversible and unaffected by prior ATPgammaS-induced channel phosphorylation. These results suggest that cocaine directly binds and facilitates the opening of L-type calcium channels. Importantly, elevated intracellular calcium levels via this mechanism triggering second messenger pathways and gene activation may contribute to many of the cardiovascular and central nervous system effects of cocaine.

Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A.

The NMDA (N-methyl-D-aspartate) subclass of glutamate receptor is essential for the synaptic plasticity thought to underlie learning and memory and for synaptic refinement during development. It is currently believed that the NMDA receptor (NMDAR) is a heteromultimeric channel comprising the ubiquitous NR1 subunit and at least one regionally localized NR2 subunit. Here we report the characterization of a regulatory NMDAR subunit, NR3A (formerly termed NMDAR-L or chi-1), which is expressed primarily during brain development. NR3A co-immunoprecipitates with receptor subunits NR1 and NR2 in cerebrocortical extracts. In single-channel recordings from Xenopus oocytes, addition of NR3A to NR1 and NR2 leads to the appearance of a smaller unitary conductance. Genetic knockout of NR3A in mice results in enhanced NMDA responses and increased dendritic spines in early postnatal cerebrocortical neurons. These data suggest that NR3A is involved in the development of synaptic elements by modulating NMDAR activity.

Stoichiometry of recombinant N-methyl-D-aspartate receptor channels inferred from single-channel current patterns.

Single-channel currents were recorded from mouse NR1-NR2B (zeta-epsilon2) receptors containing mixtures of wild-type and mutant subunits expressed in Xenopus oocytes. Mutant subunits had an asparagine-to-glutamine (N-to-Q) mutation at the N0 site of the M2 segment (NR1:598, NR2B:589). Receptors with pure N or Q NR1 and NR2 subunits generated single-channel currents with distinctive current patterns. Based on main and sublevel amplitudes, occupancy probabilities, and lifetimes, four patterns of current were identified, corresponding to receptors with the following subunit compositions (NR1/NR2): N/N, N/Q, Q/N, and Q/Q. Only one current pattern was apparent for each composition. When a mixture of N and Q NR2 subunits was coexpressed with pure mutant NR1 subunits, three single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as Q/N receptors. The third, novel pattern presumably arose from hybrid receptors having both N and Q NR2 subunits. When a mixture of N and Q NR1 subunits was coexpressed with pure mutant NR2 subunits, six single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as N/Q receptors. The four novel patterns presumably arose from hybrid receptors having both N and Q NR1 subunits. The relative frequency of NR1 hybrid receptor current patterns depended on the relative amounts of Q and N subunits that were injected into the oocytes. The number of hybrid receptor patterns suggests that there are two NR2 subunits per receptor and is consistent with either three or five NR1 subunits per receptor, depending on whether or not the order of mutant and wild-type subunits influences the current pattern. When considered in relation to other studies, the most straightforward interpretation of the results is that N-methyl-D-aspartate receptors are pentamers composed of three NR1 and two NR2 subunits.

Subconductance states of a mutant NMDA receptor channel kinetics, calcium, and voltage dependence.

The kinetic properties of main and subconductance states of a mutant mouse N-methyl-D-aspartate (NMDA) receptor channel were examined. Recombinant receptors made of zeta-epsilon 2 (NR1-NR2B) subunits having asparagine-to-glutamine mutations in the M2 segment (zeta N598Q/epsilon 2N589Q) were expressed in Xenopus oocytes. Single channel currents recorded from outside-out patches were analyzed using hidden Markov model techniques. In Ca(2+)-free solutions, an open receptor channel occupies a main conductance (93 pS) and a subconductance (62 pS) with about equal probability. There are both brief and long-lived subconductance states, but only a single main level state. At -80 mV, the lifetime of the main and the longer-lived sub level are both approximately 3.3 ms. The gating of the pore and the transition between conductance levels are essentially independent processes. Surprisingly, hyperpolarization speeds both the sub-to-main and main-to-sub transition rate constants (approximately 120 mV/e-fold change), but does not alter the equilibrium occupancies. Extracellular Ca2+ does not influence the transition rate constants. We conclude that the subconductance levels arise from fluctuations in the energetics of ion permeation through a single pore, and that the voltage dependence of these fluctuations reflects the modulation by the membrane potential of the barrier between the main and subconductance conformations of the pore.

Identification of a high affinity divalent cation binding site near the entrance of the NMDA receptor channel.

Single channel currents from recombinant N-methyl-D-aspartate (NMDA) receptors having an N-to-Q mutation in M2 reveal a divalent cation binding site that is near the entrance of the pore (approximately 0.2 through the electric field). Ca2+ rapidly binds to this site and readily permeates the channel, while Mg2+ binds more slowly and does not permeate as readily. In wild-type receptors, Mg2+ also blocks the current by occupying a site that is approximately 0.6 through the field. When the more external site is occupied by Ca2+, the conductance of the pore to NA+ is reduced but not abolished, perhaps by an electrostatic blocking mechanism. The site serves to enrich the fraction of NMDA receptor current carried by CA2+.

Ion channels formed by NB, an influenza B virus protein.

The influenza B virus protein, NB, was expressed in Escherichia coli, either with a C-terminal polyhistidine tag or with NB fused to the C-terminus of glutathione S-transferase (GST), and purified by affinity chromatography. NB produced ion channel activity when added to artificial lipid bilayers separating NaCl solutions with unequal concentrations (150-500 mM cis, 50 mM trans). An antibody to a peptide mimicking the 25 residues at the C-terminal end of NB, and amantadine at high concentration (2-3 mM), both depressed ion channel activity. Ion channels had a variable conductance, the lowest conductance observed being approximately 10 picosiemens. At a pH of 5.5 to 6.5, currents reversed at positive potentials indicating that the channel was more permeable to sodium than to chloride ions (PNa/PCl approximately 9). In asymmetrical NaCl solutions at a pH of 2.5, currents reversed closer to the chloride than to the sodium equilibrium potential indicating that the channel had become more permeable to chloride than to sodium ions (PCl/PNa approximately 4). It was concluded that, at normal pHs, NB forms cation-selective channels.

Blockade of a resting potassium channel and modulation of synaptic transmission by ecstasy in the hippocampus.

3,4-Methylenedioxymethamphetamine (ecstasy, MDMA) and related amphetamines are CNS stimulants that have euphoric, memory-enhancing and neurotoxic properties. When applied in pharmacological doses to cultured rat hippocampal neurons, ecstasy reduced the conductance of a 50-pS barium-sensitive resting K+ channel and increased neuronal excitability. Ecstasy enhanced synaptic strength by irreversibly increasing the amplitude of excitatory autaptic currents and the frequency of spontaneous excitatory postsynaptic currents. Ecstasy did not alter the amplitude of inhibitory autaptic currents or the frequency of spontaneous inhibitory postsynaptic currents but reversibly prolonged the decay phase of inhibitory autaptic currents and spontaneous inhibitory postsynaptic currents. These results suggest that K+ channel blockade and the effects on synaptic transmission may contribute to the pharmacological effects of ecstasy and other amphetamines.

Potassium channels activated by GABAB agonists and serotonin in cultured hippocampal neurons.

1. Single-channel currents were recorded in cell-attached patches on cultured hippocampal neurons in response to gamma-aminobutyric acid-B (GABAB) agonists or serotonin applied to the cell surface outside the patch area. 2. The channels activated by GABAB agonists and serotonin were potassium selective but had a different conductance and kinetic behavior. Channels activated by GABAB agonists had a higher conductance, longer open-time, and longer burst-length than channels activated by serotonin. 3. The kinetic behavior of channels activated by GABAB agonists varied with potential whereas channels activated by serotonin did not show voltage-dependent changes in kinetics. 4. In a few cell-attached patches, both types of channel were activated when the cell was exposed to GABA together with serotonin. 5. It was concluded that GABAB agonists and serotonin activate different potassium channels in the soma of cultured hippocampal neurons.

The influence of membrane potential on chloride channels activated by GABA in rat cultured hippocampal neurons.

Chloride currents were activated by a low concentration of GABA (0.5 microM) in neonatal rat hippocampal neurons cultured for up to 14 days. Currents elicited by 0.5 microM GABA in neurons, voltage-clamped using the whole-cell technique with pipettes containing 149 mM Cl-, reversed close to 0 mV whether pipettes contained 144 mM Na+ or 140 mM Cs+, and were blocked by 100 microM bicuculline. Current-voltage curves showed outward rectification. Single channel currents appeared in cell-attached patches when the pipette tip was perfused with pipette solution containing 0.5 microM GABA and disappeared when a solution containing 100 microM bicuculline plus 0.5 microM GABA was injected into the pipette tip. The channels showed outward rectification and, in some patches, had a much lower probability of opening at hyperpolarized potentials. The average chord conductance in 10 patches hyperpolarized by 80 mV was 7.8 +/- 1.6 pS (SEM) compared with a chord conductance of 34.1 +/- 3.5 pS (SEM) in the same patches depolarized by 80 mV. Similar single channel currents were also activated in cell-free, inside-out patches in symmetrical chloride solutions when 0.5 microM GABA was injected into the pipette tip. The channels showed outward rectification similar to that seen in cell-attached patches, and some channels had a lower probability of opening at hyperpolarized potentials. The average chord conductance in 13 patches hyperpolarized by 80 mV was 11.8 +/- 2.3 pS (SEM) compared with 42.1 +/- 3.1 pS (SEM) in the same patches depolarized by 80 mV.

Synthesis and pharmacological evaluation of the antifibrillatory effect of fluorinated derivatives of carazolol and celiprolol: comparison with propranolol.

1. The effects of carazolol, celiprolol and their respective fluorinated derivatives (FD) on electrical stimulation threshold (EST) and ventricular fibrillation threshold (VFT) were compared with those of propranolol and propranolol-FD in Langendorff-perfused rabbit hearts. 2. Carazolol, carazolol-FD, propranolol and propranolol-FD produced significant dose-dependent elevation of both EST and VFT at all tested concentration levels. In contrast, celiprolol and celiprolol-FD did not produce a significant change in either threshold. 3. On a dosage basis, the order of antifibrillatory potency of these compounds is: carazolol-FD > propranolol-FD > carazolol > propranolol > celiprolol-FD > celiprolol. 4. The results of this study seem to indicate the importance of membrane stabilizing effect for a potent antifibrillatory action of beta-adrenoceptor blocking agents. Furthermore, fluorination is demonstrated to produce more potent antifibrillatory activity than that of the parent drugs.

Coupled potassium channels induced by arachidonic acid in cultured neurons.

Exposure of the inside surface of patches of membrane excised from cultured rat hippocampal neurons to arachidonic acid (10-100 microM) caused the appearance of potassium currents of variable amplitude similar to those activated by GABA or baclofen in cell-attached patches. The amplitude of single-channel currents increased with time after exposure to 20 or 50 microM arachidonic acid and also increased when arachidonic acid concentration was increased from 20 to 50 or 100 microM. Current-amplitude probability histograms had peaks at integral multiples of an 'elementary' current. It is proposed that arachidonic acid or its metabolites cause synchronous opening and closing of coupled conducting units (co-channels) in cell membranes.

Characterization of single channel currents using digital signal processing techniques based on Hidden Markov Models.

Techniques for extracting small, single channel ion currents from background noise are described and tested. It is assumed that single channel currents are generated by a first-order, finite-state, discrete-time, Markov process to which is added 'white' background noise from the recording apparatus (electrode, amplifiers, etc). Given the observations and the statistics of the background noise, the techniques described here yield a posteriori estimates of the most likely signal statistics, including the Markov model state transition probabilities, duration (open- and closed-time) probabilities, histograms, signal levels, and the most likely state sequence. Using variations of several algorithms previously developed for solving digital estimation problems, we have demonstrated that: (1) artificial, small, first-order, finite-state, Markov model signals embedded in simulated noise can be extracted with a high degree of accuracy, (2) processing can detect signals that do not conform to a first-order Markov model but the method is less accurate when the background noise is not white, and (3) the techniques can be used to extract from the baseline noise single channel currents in neuronal membranes. Some studies have been included to test the validity of assuming a first-order Markov model for biological signals. This method can be used to obtain directly from digitized data, channel characteristics such as amplitude distributions, transition matrices and open- and closed-time durations.

GABA-induced potassium channels in cultured neurons.

When gamma-aminobutyric acid (GABA) or baclofen were applied to cultured rat hippocampal neurons, single-channel potassium currents appeared after a delay of 30 s or more in patches of membrane on the cell surface isolated from the agonists by the recording pipette. The appearance of currents in patches not exposed to agonist, the delay in their appearance and the suppression of currents in cells pre-incubated with pertussis toxin indicate the involvement of an intracellular second messenger system. The channels were associated with a GABAB receptor rather than a GABAA receptor as they were blocked by baclofen, a GABAB antagonist, but were not affected by bicuculline, a GABAA antagonist. A feature of the single channel currents was their variable amplitude: they had a maximum conductance of ca. 70 pS and displayed many lower conductance states that were integral multiples of 5-6 pS. In several cells exposed to GABA or baclofen, first small currents and then progressively larger currents appeared: current amplitude was a multiple of an elementary current. It is suggested that binding of GABA to GABAB receptors activates a second messenger system causing opening of oligomeric potassium channels.

Evaluation of the antifibrillatory activity of fluorinated derivatives of indenolol and nadolol in isolated rabbit and rat hearts: comparison with propranolol.

The effects of indenolol and nadolol and of their respective fluorinated derivatives on ventricular fibrillation threshold were compared with propranolol in Langendorff-perfused rabbit hearts. Their effects on aconitine-induced ventricular tachycardia and fibrillation were also evaluated in Langendorff-perfused rat hearts. Propranolol, indenolol and indenolol fluorinated derivative produced significant dose-dependent increases in ventricular fibrillation threshold at all tested concentration levels. These compounds have also caused significant increases in the amount of aconitine required to produce ventricular tachycardia and fibrillation. Nadolol and nadolol fluorinated derivative did not produce any significant changes in the above-mentioned parameters except a small, but statistically significant, increase in ventricular fibrillation threshold produced by the highest concentration (2.64 microM) tested of nadolol fluorinated derivative. On a dosage basis, the order of potency of these compounds is indenolol fluorinated derivative greater than propranolol greater than indenolol greater than nadolol fluorinated derivative greater than nadolol. These results suggest that additional properties of beta-adrenergic blockers, such as membrane-stabilizing effects, may contribute to their antifibrillatory activity and that fluorination may tend to increase it.