Maxi-K potassium channels: form, function, and modulation of a class of endogenous regulators of intracellular calcium.

Large-conductance calcium-activated (maxi-K, BK) potassium channels are widely distributed in the brain. Maxi-K channels function as neuronal calcium sensors and contribute to the control of cellular excitability and the regulation of neurotransmitter release. Little is currently known of any significant role of maxi-K channels in the genesis of neurological disease. Recent advances in the molecular biology and pharmacology of these channels have revealed sources of phenotypic variability and demonstrated that they can be successfully modulated by pharmacological agents. A potential role is suggested in the treatment of conditions such as ischemic stroke and cognitive disorders.

Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels.

During ischemic stroke, neurons at risk are exposed to pathologically high levels of intracellular calcium (Ca++), initiating a fatal biochemical cascade. To protect these neurons, we have developed openers of large-conductance, Ca++-activated (maxi-K or BK) potassium channels, thereby augmenting an endogenous mechanism for regulating Ca++ entry and membrane potential. The novel fluoro-oxindoles BMS-204352 and racemic compound 1 are potent, effective and uniquely Ca++-sensitive openers of maxi-K channels. In rat models of permanent large-vessel stroke, BMS-204352 provided significant levels of cortical neuroprotection when administered two hours after the onset of occlusion, but had no effects on blood pressure or cerebral blood flow. This novel approach may restrict Ca++ entry in neurons at risk while having minimal side effects.

Differential effects of coadministration of fluoxetine and WAY-100635 on serotonergic neurotransmission in vivo: sensitivity to sequence of injections.

Serotonin 5-HT(1A) receptor antagonists potentiate the effects of serotonin reuptake inhibitors on extracellular serotonin levels in a variety of brain regions. These effects are quite variable, however, with reports indicating potentiations of anywhere from 100-1900%. One factor that might impact the magnitude of such potentiations is the timing of administration of the two agents; reports in which the reuptake inhibitor is given prior to the serotonin receptor antagonist consistently report larger potentiations than reports in which the antagonist is given first. To test this relationship directly, microdialysis and electrophysiology studies were performed to assess the magnitude of increase in extracellular serotonin and changes in cellular activity produced by the serotonin reuptake inhibitor fluoxetine and the 5-HT(1A) receptor antagonist WAY-100635 under various dosing regimens. In microdialysis studies, when WAY-100635 (0.5 mg/kg s.c.) was administered 80 min after fluoxetine (10 mg/kg i.p.) the increase in serotonin was more than twice that observed when the compounds were coadministered. In electrophysiology studies in vivo, WAY-100635 reversed the depression of cell firing produced by fluoxetine when administered 30 min after fluoxetine, but when the two compounds were coadministered, a depression in firing rate was observed comparable to that produced by fluoxetine alone. In contrast, slice recording studies showed that WAY-100635 blocked the effects of fluoxetine regardless of the order of administration. These results indicate that fluoxetine and WAY-100635 can interact in a fashion not predicted by the currently accepted model. It is likely that neuronal circuitry outside of the raphe nuclei underlies this relationship.

The augmentation hypothesis for improvement of antidepressant therapy: is pindolol a suitable candidate for testing the ability of 5HT1A receptor antagonists to enhance SSRI efficacy and onset latency?

The development of selective serotonin reuptake inhibitors (SSRIs) provided a major advancement in the treatment of depression. However, these drugs suffer from a variety of drawbacks, most notably a delay in the onset of efficacy. One hypothesis suggests that this delay in efficacy is due to a paradoxical decrease in serotonergic (5-HT) neuronal impulse flow and release, following activation of inhibitory presynaptic 5-HT1A autoreceptors, following acute administration of SSRIs. According to the hypothesis, efficacy is seen only when this impulse flow is restored following desensitization of 5-HT1A autoreceptors and coincident increases in postsynaptic 5-HT levels are achieved. Clinical proof of this principal has been suggested in studies that found a significant augmenting effect when the beta-adrenergic/5-HT1A receptor antagonist, pindolol, was coadministered with SSRI treatment. In this article, we review preclinical electrophysiological and microdialysis studies that have examined this desensitization hypothesis. We further discuss clinical studies that utilized pindolol as a test of this hypothesis in depressed patients and examine preclinical studies that challenge the notion that the beneficial effect of pindolol is due to functional antagonism of the 5-HT1A autoreceptors.

Cognition-enhancing drugs increase stimulated hippocampal theta rhythm amplitude in the urethane-anesthetized rat.

Synchronous hippocampal electroencephalographic activity occurring in a frequency range of 3 to12 Hz (i.e., hippocampal theta rhythm) has been associated with mnemonic processes in vivo. However, this link is tenuous and theta rhythm may be secondary to processes that underlie mnemonic function. If theta rhythm is associated with mnemonic or cognitive function, cognition-enhancing drugs should enhance theta rhythm regardless of their primary biological target. In the current study, we evaluated several drugs that were shown to have cognition-enhancing properties in preclinical behavioral models and that vary with respect to their primary biological target: 1) the nootropic piracetam (250 and 500 mg/kg); 2) the small-conductance calcium-activated potassium-channel blocker apamin (0.1 and 0.4 mg/kg); and 3) the acetylcholinesterase inhibitor donepezil (0.1-10.0 mg/kg). All of the cognition-enhancing drugs produced dose-dependent increases in hippocampal theta rhythm amplitude elicited by stimulation of the brainstem reticular formation at doses that did not affect peak theta frequency in the urethane-anesthetized rat. These increases were reversed by the muscarinic receptor antagonist scopolamine, suggesting a common final cholinergic action of these compounds. The use-dependent N-methyl-D-aspartate antagonist dizocilipine maleate and scopolamine reduced theta amplitude (both) and frequency (dizocilipine maleate only). These data demonstrate that hippocampal theta rhythm is sensitive to cognition-modulating compounds, suggesting that theta rhythm may be closely associated with cognitive function.

A reexamination of the role of GABA in the mammalian suprachiasmatic nucleus.

Three independent electrophysiological approaches in hypothalamic slices were used to test the hypothesis that gamma-amino butyric acid (GABA)A receptor activation excites suprachiasmatic nucleus (SCN) neurons during the subjective day, consistent with a recent report. First, multiple-unit recordings during either the subjective day or night showed that GABA or muscimol inhibited firing activity of the SCN population in a dose-dependent manner. Second, cell-attached recordings during the subjective day demonstrated an inhibitory effect of bath- or microapplied GABA on action currents of single SCN neurons. Third, gramicidin perforated-patch recordings showed that bicuculline increased the spontaneous firing rate during the subjective day. Therefore, electrophysiological data obtained by three different experimental methods provide evidence that GABA is inhibitory rather than excitatory during the subjective day.

An assessment of the present and future roles of non-ligand gated ion channel modulators as CNS therapeutics.

Few approved drugs have, as their primary known mechanism of action, modulation of non-ligand gated ion channels. However, these proteins are important regulators of neuronal function through their control of sodium, potassium, calcium and chloride flux, and are ideal candidates as drug discovery targets. Recent progress in the molecular biology and pharmacology of ion channels suggests that many will be associated with specific pharmacological profiles that will include both activators and inhibitors. Ion channels, through their regulation by G-proteins, are a major component of the final common pathway of many drugs acting at classical neuronal receptors. Thus, targeting of the ion channels themselves may confer different profiles of efficacy and specificity to drug action in the brain and spinal cord. Three areas for drug discovery are profiled that the authors consider prime targets for ion channel based therapies, anticonvulsant drugs, cognition enhancing drugs and drugs for improving neurone survival following ischaemia.

Phase shifting of circadian rhythms and depression of neuronal activity in the rat suprachiasmatic nucleus by neuropeptide Y: mediation by different receptor subtypes.

Neuropeptide Y (NPY) has been implicated in the phase shifting of circadian rhythms in the hypothalamic suprachiasmatic nucleus (SCN). Using long-term, multiple-neuron recordings, we examined the direct effects and phase-shifting properties of NPY application in rat SCN slices in vitro (n = 453). Application of NPY and peptide YY to SCN slices at circadian time (CT) 7.5-8.5 produced concentration-dependent, reversible inhibition of cell firing and a subsequent significant phase advance. Several lines of evidence indicated that these two effects of NPY were mediated by different receptors. NPY-induced inhibition and phase shifting had different concentration-response relationships and very different phase-response relationships. NPY-induced phase advances, but not inhibition, were blocked by the GABAA antagonist bicuculline, suggesting that NPY-mediated modulation of GABA may be an underlying mechanism whereby NPY phase shifts the circadian clock. Application of the Y2 receptor agonists NPY 13-36 and (Cys2,8-aminooctanoic acid5,24,D-Cys27)-NPY advanced the peak of the circadian rhythm but did not inhibit cell firing. The Y1 and Y5 agonist [Leu31,Pro34]-NPY evoked a substantial inhibition of discharge but did not generate a phase shift. NPY-induced inhibition was not blocked by the specific Y1 antagonist BIBP-3226; the antagonist also had no effect on the timing of the peak of the circadian rhythm. Application of the Y5 agonist [D-Trp32]-NPY produced only direct neuronal inhibition. These are the first data to indicate that at least two functional populations of NPY receptors exist in the SCN, distinguishable on the basis of pharmacology, each mediating a different physiological response to NPY application.

Formation of intermediate-conductance calcium-activated potassium channels by interaction of Slack and Slo subunits.

Large-conductance calcium-activated potassium channels (maxi-K channels) have an essential role in the control of excitability and secretion. Only one gene Slo is known to encode maxi-K channels, which are sensitive to both membrane potential and intracellular calcium. We have isolated a potassium channel gene called Slack that is abundantly expressed in the nervous system. Slack channels rectify outwardly with a unitary conductance of about 25-65 pS and are inhibited by intracellular calcium. However, when Slack is co-expressed with Slo, channels with pharmacological properties and single-channel conductances that do not match either Slack or Slo are formed. The Slack/Slo channels have intermediate conductances of about 60-180 pS and are activated by cytoplasmic calcium. Our findings indicate that some intermediate-conductance channels in the nervous system may result from an interaction between Slack and Slo channel subunits.

Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock.

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.

Use of subunit-specific antisense oligodeoxynucleotides to define developmental changes in the properties of N-methyl-D-aspartate receptors.

Antisense oligodeoxynucleotides were used to determine whether alterations in the expression of N-methyl-D-aspartate (NMDA) receptor subunit mRNA are responsible for developmental changes in the sensitivity of receptors to agonists and antagonists. Xenopus laevis oocytes were injected with mRNA prepared from neonatal and adult rat cerebral cortex, and the effects of agonists and antagonists were determined under voltage-clamp conditions. Glycine-site antagonists like 7-chlorokynurenate and glutamate-site antagonists like CGP-39653 were more potent at NMDA receptors expressed from mRNA from adult rat cerebral cortex than those expressed from mRNA from 1-day-old rat. NMDA receptors from 1-day-old rat cerebral cortex were more sensitive to activation by glycine than were receptors from adult rat cerebral cortex. 7-Chlorokynurenate and CGP-39653 were more potent inhibitors of responses seen with heteromeric NR1/NR2A receptors than with NR1/ NR2B receptors. Conversely, heteromeric NR1/NR2B receptors were more sensitive to activation by glycine than were NR1/NR2A receptors. We previously described a delay in the expression of the NR2A subunit in developing rat brain. Anti-sense oligodeoxynucleotides were used to determine whether the delayed expression of the NR2A subunit underlies changes in pharmacological properties observed during development. The properties of receptors seen when adult brain mRNA was coinjected with antisense oligodeoxynucleotides against the NR2A subunit were similar to those found in receptors from 1-day-old rat brain. These data suggest that changes in the sensitivity of NMDA receptors to antagonists and to glycine seen during development are a result of alterations in the expression of different species of NR2 subunit mRNA.

Synthesis and biological evaluation of an iodinated iberiotoxin analogue, [mono-iodo-Tyr5, Phe36]-iberiotoxin.

The synthesis and iodination of a structural analogue of the specific large-conductance calcium-activated potassium (BK) channel blocker, iberiotoxin (IbTX), a 37-amino acid scorpion neurotoxin, is reported. The synthesis of this analogue, [Tyr5, Phe36]-IbTX, was accomplished using standard solid-phase Fmoc (9-fluorenylmethoxycarbonyl) chemistry protocols. The linear peptide was cyclized via the formation of three intramolecular disulfide bridges and subsequently iodinated at the Tyr5 position. Upon purification, the iodinated analogue, [mono-iodo-Tyr5, Phe36]-IbTX, exhibited comparable biological activity to native IbTX in blocking BK-mediated currents. These findings suggest the synthesis and use of an 125I labelled IbTX analogue for BK channel localization in autoradiography experiments.

Phenotypic alteration of a human BK (hSlo) channel by hSlobeta subunit coexpression: changes in blocker sensitivity, activation/relaxation and inactivation kinetics, and protein kinase A modulation.

A human homolog of the large-conductance calcium-activated potassium (BK) channel beta subunit (hSlobeta) was cloned, and its effects on a human BK channel (hSlo) phenotype are reported. Coexpression of hSlo and hSlobeta, in both oocytes and human embryonic kidney 293 cells, resulted in increased Ca2+ sensitivity, marked slowing of BK channel activation and relaxation, and significant reduction in slow inactivation. In addition, coexpression changed the pharmacology of the BK channel phenotype: hSlo-mediated currents in oocytes were more sensitive to the peptide toxin iberiotoxin than were hSlo + hSlobeta currents, and the potency of blockade by the alkaloid BK blocker tetrandrine was much greater on hSlo + hSlobeta- mediated currents compared with hSlo currents alone. No significant differences in the response to charybdotoxin or the BK channel opener NS1619 were observed. Modulation of BK channel activity by phosphorylation was also affected by the presence of the hSlobeta subunit. Application of cAMP-dependent protein kinase increased P(OPEN) of hSlo channels, but decreased P(OPEN)of most hSlo + hSlobeta channels. Taken together, these altered characteristics may explain some of the wide diversity of BK channel phenotypes observed in native tissues.

Effects of channel modulators on cloned large-conductance calcium-activated potassium channels.

Through expression of the cloned mouse (mSlo) or human (hSlo) large-conductance (BK) Ca(2+)-activated K+ channel in Xenopus laevis oocytes and HEK 293 cells, we characterized the effects of reported blockers and openers of BK channels to initiate the study of the molecular determinants of BK channel modulation. In oocytes, iberiotoxin and charybdotoxin, peptidyl scorpion toxins, were both equally effective blockers of BK current, although iberiotoxin was significantly more potent than charybdotoxin. The structurally related peptide kaliotoxin was not a potent blocker of BK current. Paxilline, a fungal tremorgenic alkaloid, was an effective but complex blocker of BK current. Tetrandrine, a putative blocker of type II BK channels, and ketamine were relatively ineffective. The putative BK openers NS004 and NS1619, phloretin, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) increased BK current in oocytes at microM concentrations; many of these produced biphasic concentration-response relationships. Coapplication of representative blockers and openers revealed several patterns of interaction, including competitive and noncompetitive antagonism. NS1619, niflumic acid, and phloretin were tested by using excised inside-out membrane patches from HEK 293 cells and were found to increase the activity of hSlo BK channels and produce a leftward shift in the G/Gmax-versus-voltage relationship of these channels. These results represent the first comprehensive examination of the molecular pharmacology of BK channels.

Reduced electrical excitability of PC12 cells deficient in GAP-43: comparison with GAP-43-positive cells.

The electrical excitability of 3 lines of rat pheochromocytoma (PC12) cells were determined under current-clamp recording conditions. In the presence of nerve growth factor (NGF), PC12(A) 'control' cells expressed high levels of GAP-43 protein, PC12(B) cells were highly deficient in GAP-43, and PC12(AB) cells, created by transfection of PC12(B) cells with a rat GAP-43 gene construct, expressed high levels of GAP-43. All 3 lines had similar resting membrane potentials, but significantly greater proportions of GAP-43-containing PC12(A) and PC12(AB) cells exhibited spiking in response to depolarizing current pulses. These spikes were resistant to TTX, were greatly enhanced in TEA and TTX, and were substantially reduced by L-type Ca(2+)-channel antagonists. GAP-43 expression may regulate PC12 cell excitability following NGF treatment, as reflected in a lower proportion of cells capable of discharging with Ca(2+)-spikes in a GAP-43-deficient cell line.

Cloning and expression of a human large-conductance calcium-activated potassium channel.

A putative BK channel gene was cloned from a human brain (substantia nigra) cDNA library by hybridization screening. The sequence of the full length clone shows high homology with the mSlo gene, suggesting that this cDNA is the human homologue (hSlo). The hSlo clone does not contain either alternative exon A or B at its splice sites; and similar to mSlo, it has a long string of serines at its 5' end. Reverse transcription coupled with the PCR technique demonstrated the differences in expression of the isoforms among the CNS and the periphery. Expression of hSlo in Xenopus oocytes showed a family of outward currents, induced by step depolarizations, that were blocked by iberiotoxin and activated by the compound NS004, a known opener of native and cloned maxi-K channels. Single channel recordings of hSlo channels showed a high degree of voltage- and Ca(2+)-dependence, and an average single channel conductance of 285.9 pS.

Reduced bicuculline response and GABAA agonist binding in aged rat hippocampus.

Extracellular field recordings from CA1 pyramidal cells in the rat hippocampal slice preparation were used to examine the effects of age on gamma-aminobutyric acid (GABA)-mediated recurrent inhibition. The actions of bicuculline (1-100 microM), a GABAA antagonist, were assessed in slices from young (1-3 months) and aged (26 months) Fischer 344 rats. Pre-drug population spike amplitudes were smaller in slices from aged rats. Bicuculline increased population spike amplitudes in slices from both age groups, but slices from young rats were more sensitive to the antagonist. Bicuculline also produced multiple population spikes in slices from both age groups, however the increase in population spike burst durations was much greater in slices from young rats than in slices from aged rats. Agonist radiolabeled GABAA binding site density was significantly decreased in hippocampal tissue from aged rats. Our results suggest there is a reduction in GABAergic inhibition in hippocampal slices from aged rats, possibly mediated by a decrease in GABAA receptors.

Opening of large-conductance calcium-activated potassium channels by the substituted benzimidazolone NS004.

1. We used electrophysiological techniques to examine the effects of 5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidaz ole- 2-one (NS004) on large-conductance calcium-activated potassium (BK) channels. 2. We used recordings from excised membrane patches (cell-attached and inside-out single-channel configurations) and whole-cell patch-clamp recordings to examine the effects of NS004 on single BK channels and whole-cell outward currents, respectively, in rat GH3 clonal pituitary tumor cells. We also tested NS004 on voltage-clamped BK channels isolated from rat brain plasma membrane preparations and reconstituted into planar lipid bilayers. Finally, we used two-electrode voltage-clamp techniques to study the effects of NS004 on currents expressed in Xenopus laevis oocytes by the recently described Slo BK clone from Drosophila. 3. In GH3 cells and in Xenopus oocytes expressing the Slo gene product NS004 produced an increase in an iberiotoxin- or tetraethylammonium-sensitive whole-cell outward current, respectively. NS004 produced a significant increase in the activity of single GH3 cell BK channels and rat brain BK channels reconstituted into planar lipid bilayers. In both systems this was characterized by an increase in channel mean open time, a decrease in interburst interval, and an apparent increase in channel voltage/calcium sensitivity. 4. These data indicate that NS004 could be useful for investigating the biophysical and molecular properties of BK channels and for determining the functional consequences of the opening of BK channels.

The substituted benzimidazolone NS004 is an opener of the cystic fibrosis chloride channel.

Cystic fibrosis is a major inherited disorder involving abnormalities of fluid and electrolyte transport in a number of different organs. Epithelial cells of cystic fibrosis patients have a decreased capacity to secrete chloride in response to cAMP-mobilizing agents because of the mutation of a single gene. The gene product, the cystic fibrosis transmembrane conductance regulator or CFTR, is a chloride channel. The most frequent mutation is a deletion of phenylalanine in position 508 (delta F508-CFTR) that reduces both the expression of the CFTR protein at the cell surface, and the activity of the Cl- channel. This work presents the properties of NS004, a substituted benzimidazolone, which is the first activator of normal and mutant CFTR-associated chloride channels to be described. NS004 activated CFTR and delta F508-CFTR Cl- channels expressed in Xenopus oocytes, and increased 125I efflux (via the Cl- channel) from Vero cells expressing CFTR and delta F508-CFTR. Application of NS004 to the external side of outside-out patches excised from these CFTR- and delta F508-CFTR-expressing cells induced a marked and reversible increase in channel activity.