Structure and functional characterization of a novel human low-voltage activated calcium channel.

We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca2+ channel alpha1 subunit, alpha1H, from a human medullary thyroid carcinoma cell line. The alpha1H subunit is structurally similar to previously described alpha1 subunits. Northern blot analysis indicates that alpha1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba2+ currents recorded from human embryonic kidney 293 cells transiently expressing alpha1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (tau = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing alpha1H. Single-channel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of approximately 9 pS. These channels are blocked by Ni2+ (IC50 = 6.6 microM) and the T-type channel antagonists mibefradil (approximately 50% block at 1 microM) and amiloride (IC50 = 167 microM). Thus, alpha1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca2+ channels.

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents.

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than nonspecific agents such as nicotine.

Phenylethanolamines inhibit NMDA receptors by enhancing proton inhibition.

The phenylethanolamines, ifenprodil and CP-101,606, are NMDA receptor antagonists with promising neuroprotective properties. In recombinant NMDA receptors expressed in Xenopus oocytes, we found that these drugs inhibit NMDA receptors through a unique mechanism, making the receptor more sensitive to inhibition by protons, an endogenous negative modulator. These findings support a critical role for the proton sensor in gating the NMDA receptor and point the way to identifying a context-dependent NMDA receptor antagonist that is inactive at physiological pH, but is a potent inhibitor during the acidic conditions that arise during epilepsy, ischemia and brain trauma.

Conformationally restricted analogues of nicotine and anabasine.

A series of conformationally restricted analogues of nicotine has been synthesized and evaluated as agonists of neuronal acetylcholine receptors. Compound 2 (SIB-1663), which selectively activated human recombinant alpha 2 beta 4 and alpha 4 beta 4 nAChRs, was shown to be active in animal models of Parkinson's disease and pain.

Pharmacological characterization of recombinant human neuronal nicotinic acetylcholine receptors h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7 expressed in Xenopus oocytes.

Human neuronal nicotinic acetylcholine receptors (nAChRs) h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7 were expressed in Xenopus oocytes and tested for their sensitivities to the nicotinic agonists acetylcholine (ACh), nicotine, cytisine (CYT) and 1,1-dimethyl-4-phenylpiperazinium (DMPP) and the nAChR. antagonists mecamylamine (MEC), d-tubocurarine and dihydro-beta-erythroidine. CYT was the least efficacious agonist at hnAChRs containing beta 2 subunits, but it displayed significant activity at h alpha 2 beta 4, h alpha 3 beta 4, h alpha 4 beta 4 and h alpha 7 nAChRs. ACh was one of the most efficacious agonists at all hnAChRs, except at h alpha 3 beta 2, where DMPP was markedly more efficacious than ACh. ACh was among the least potent agonists at all hnAChRs. The rank order of potency displayed by h alpha 3 beta 2 and h alpha 3 beta 4 nAChRs (DMPP approximately CYT approximately nicotine > ACh and DMPP > CYT approximately nicotine > ACh, respectively), differs from that reported for their rat homologs (Luetje and Patrick, 1991; Covernton et al., 1994). The agonist profile observed in h alpha 7 also differs from that reported for its rat homolog (Seguela et al., 1993). Human alpha 4 beta 2 and h alpha 4 beta 4 nAChRs were more sensitive to dihydro-beta-erythroidine than d-tubocurarine, whereas h alpha 7 and h alpha 3 beta 4 were more sensitive to d-tubocurarine than dihydro-beta-erythroidine. These antagonists were equipotent at h alpha 2 beta 2, h alpha 3 beta 2 and h alpha 2 beta 4 nAChRs. MEC (3 microM) inhibited h alpha 2 beta 4 and h alpha 4 beta 4 nAChRs by > 80%, whereas h alpha 2 beta 2, h alpha 4 beta 2 and h alpha 7 nAChRs were inhibited by approximately 50%. Taken together, the differential sensitivities observed at various recombinant hnAChR subtypes indicate that both alpha and beta subunits contribute to the pharmacology of these ligand-gated channels. The unique selectivity profiles displayed by human nAChRs constitute a valuable tool for the development of selective nicotinic analogs as potential therapeutic drugs.

Differential dependence on GluR2 expression of three characteristic features of AMPA receptors.

The GluR2 subunit controls three key features of ion flux through the AMPA subtype of glutamate receptors-calcium permeability, inward rectification, and channel block by external polyamines, but whether each of these features is equally sensitive to GluR2 abundance is unknown. The relations among these properties were compared in native AMPA receptors expressed by acutely isolated hippocampal interneurons and in recombinant receptors expressed by Xenopus oocytes. The shape of current-voltage (I-V) relations between -100 and +50 mV for either recombinant or native AMPA receptors was well described by a Woodhull block model in which the affinity for internal polyamine varied over a 1000-fold range in different cells. In oocytes injected with mixtures of GluR2:non-GluR2 mRNA, the relative abundance of GluR2 required to reduce the log of internal blocker affinity by 50% was two- to fourfold higher than that needed to half-maximally reduce divalent permeability or channel block by external polyamines. Likewise, in interneurons the affinity of externally applied argiotoxin for its blocking site was a steep function of internal blocker affinity. These results indicate that the number of GluR2 subunits in AMPA receptors is variable in both oocytes and interneurons. More GluR2 subunits in an AMPA receptor are required to maximally reduce internal blocker affinity than to abolish calcium permeability or external polyamine channel block. Accordingly, single-cell RT-PCR showed that approximately one-half of the physiologically characterized interneurons exhibiting inwardly rectifying AMPA receptors expressed detectable levels of edited GluR2. The physiological effects of a moderate change in GluR2 relative abundance, such as occurs after ischemia or seizures or after chronic exposure to morphine, thus will be dependent on the ambient GluR2 level in a cell-specific manner.

Block of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by polyamines and polyamine toxins.

A variety of polyamine spider and wasp toxins are known to block N-methyl-D-aspartate receptor channels and recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the edited glutamate receptor (GluR)2 subunit. Recently, inward rectification of GluR2-lacking AMPA receptors was shown to be caused by voltage-dependent block by intracellular spermine. Here we demonstrate that, when applied extracellularly, the endogenous polyamines spermine and spermidine, as well as monoacylated spermine analogs and the polyamine toxins ageltoxin-489 and philanthotoxin-433, exerted a use-dependent and weakly voltage-dependent block of AMPA receptors that lack the edited GluR2 subunit, when the recombinant receptors were expressed in Xenopus oocytes. External spermine and polyamine toxins were also effective blockers of AMPA receptor mutants that did not not show inwardly rectifying kainate responses but had high calcium permeability. The polyamines and polyamine toxins also markedly reduced inwardly rectifying currents of native AMPA receptors expressed by a class of hippocampal interneurons in rat CA3 stratum radiatum that appear not to express the GluR2 subunit. In contrast, polyamines had little or no effect on the linear or outwardly rectifying kainate responses of other interneurons or CA3 pyramidal cells in which GluR2 mRNA was routinely detected. Together with previous reports, these data suggest that endogenous polyamines may bind to GluR2-lacking AMPA receptors at two or more distinct sites, one near the cytoplasmic side of the pore and the other nearer the outer side of the pore.

Nerve growth factor facilitates cholinergic neurotransmission between nucleus basalis and the amygdala in rat: an electrophysiological analysis.

Treatment of rats in vivo with NGF promotes the survival and enhances the neurotransmitter phenotype of basal forebrain cholinergic neurons. We showed recently (Williams et al., 1993) that NGF-induced stimulations of the cholinergic markers ChAT and high-affinity choline uptake are reflected in an enhanced synthesis and release of ACh in terminals fields of basal forebrain cholinergic neurons. The objective of the present study was to determine whether such effects translate into an enhancement in neurotransmission between nucleus basalis neurons and postsynaptic target cells, and therefore are likely to be of physiological significance. Changes in cholinergic neurotransmission after NGF were assessed by comparing the ability of cholinergic pathway activation, produced by electrical stimulation of nucleus basalis or the external capsule, to elicit intracellularly recorded muscarinic responses in basolateral amygdaloid (BLA) neurons in ventral forebrain slice preparations from NGF-treated and control Fischer 344 adult rats. Chronic infusion of NGF for 3 weeks (1.2 micrograms/d, i.c.v.) increased the likelihood of eliciting cholinergic slow depolarizations (slow EPSP) via stimulation of cholinergic pathways in the slice. In addition, the frequency-response curves for generation of the cholinergic slow EPSP by nucleus basalis or external capsule stimulation were shifted approximately twofold to the left and the EF50 values significantly reduced in neurons from NGF-treated slices, compared to those in preparations from vehicle-treated or untreated controls. Treatment with NGF also resulted in a leftward shift in the frequency-response curve for cholinergic pathway-induced blockade of the slow afterhyperpolarization, without change in the maximal inhibitory effect. The NGF-induced enhancement in cholinergic synaptic effectiveness was not accompanied by alterations in the resting membrane properties or intrinsic excitability of BLA pyramidal neurons. Nor did treatment with NGF affect their chemosensitivity or responsiveness to direct postsynaptic applications of the cholinergic carbachol. We conclude from these results that chronic administration of exogenous NGF can facilitate neurotransmission within basal forebrain cholinergic projections in normal adult brain, presumably as a consequence of its ability to stimulate presynaptic mechanisms involved in synthesis and/or release of ACh.

Electrophysiological and morphological properties of rat basolateral amygdaloid neurons in vitro.

Electrophysiological and morphological properties of neurons in the rat basolateral amygdala (BLA) were assessed using intracellular recordings in brain slice preparations. The vast majority of cells studied were identified as pyramidal cells on the basis of their accommodation response and by a prominent afterhyperpolarization that followed a current-evoked burst of action potentials. The second class of cells consisted of late-firing neurons that were distinguished electrophysiologically by their very negative resting membrane potential (-82 mV) and conspicuous delay in the onset of spike firing in response to depolarizing current injection. The third class of cells, termed fast-firing neurons, possessed many of the features of intrinsic inhibitory interneurons found elsewhere in the brain. These included very brief action potentials (0.7 msec), a relatively depolarized resting membrane potential (-62 mV), and spontaneous firing at a high rate and the absence of spike frequency accommodation. Intracellular labeling with Lucifer yellow of electrophysiologically identified pyramidal and late-firing cells showed them to have pyramidal to stellate cells bodies and spine-covered dendrites. Although having an overall pyramidal-like morphology, late-firing neurons possessed cells bodies and dendritic fields that were smaller than those of pyramidal cells. Lucifer yellow-labeled fast-firing neurons had a nonpyramidal morphology, with somata that were spherical to multipolar in shape and spine-sparse or aspiny dendrites. The morphological features of these cells corresponded closely to those of GABA-containing interneurons that have been described previously in the rat BLA using immunohistochemical techniques (McDonald, 1985b). Thus, it seems likely that activation of fast-firing neurons underlies inhibitory synaptic events that are recorded in the rat BLA. Our data support the conclusion derived from previous anatomical studies that pyramidal neurons constitute the predominant cell type in the BLA and function as projection neurons in this region of the amygdala. The determination of whether late-firing cells constitute a unique class of projection neurons distinct from pyramidal cells must await the outcome of studies in which the anatomical terminations of this cell type are specified.

Inhibitory responses of rat basolateral amygdaloid neurons recorded in vitro.

The purpose of the present study was to characterize the ionic and pharmacological basis of the actions of synaptically released and exogenously applied GABA in basolateral amygdaloid pyramidal cells in vitro. Stimulation of forebrain afferents to pyramidal neurons in the basolateral amygdala evoked an excitatory postsynaptic potential followed by early and late inhibitory postsynaptic potentials. The early inhibitory postsynaptic potential had a reversal potential near -70 mV, was sensitive to changes in the chloride gradient across the membrane and was blocked by the GABAA antagonists picrotoxin and bicuculline methiodide but not by the GABAB antagonists phaclofen or 2-hydroxysaclofen. In contrast, the late inhibitory postsynaptic potential had a reversal potential of approximately -95 mV and was markedly reduced or abolished by GABAB antagonists. Pressure application of GABA to the surface of the slice typically elicited a triphasic response in basolateral amygdaloid pyramidal neurons consisting of a short-latency hyperpolarization that preceded or was superimposed on a membrane depolarization followed by a longer latency hyperpolarization. Each of the responses was associated with an increase in membrane conductance. Determinations of the reversal potential, ionic dependency and sensitivity to pharmacological blockade of each component of the GABA-induced response revealed that the initial hyperpolarizing (Erev approximately -70 mV) and depolarizing (Erev approximately -55 mV) responses were mediated by a GABAA-mediated increase in chloride conductance, whereas the late hyperpolarizing response (Erev approximately -82 mV) to GABA arose from a GABAB-mediated increase in potassium conductance. Experiments in which GABA was applied at various locations on the cell suggested that the short-latency hyperpolarization resulted from activation of somatic GABA receptors, whereas the depolarizing and late hyperpolarizing responses were generated primarily in the dendrites. In contrast to the complex membrane response profile elicited by GABA, pressure ejection of the GABAB agonist baclofen produced only membrane hyperpolarizations. Taken together, these results suggest that inhibitory responses that are recorded in basolateral amygdaloid pyramidal cells are mediated by activation of both GABAA and GABAB receptors. Consistent with findings elsewhere in the CNS, the early inhibitory postsynaptic potential and initial hyperpolarization and depolarizing response to local GABA application appear to involve a GABAA-mediated increase in chloride conductance, whereas the late inhibitory postsynaptic potential and the late hyperpolarizing response to GABA arise from a GABAB-mediated increase in potassium conductance.

Muscarinic responses of rat basolateral amygdaloid neurons recorded in vitro.

1. Intracellular recordings were obtained from pyramidal-type neurons in the basolateral amygdaloid nucleus (BLA) in slices of rat ventral forebrain and used to compare the actions of exogenously applied cholinomimetics to the effects produced by electrical stimulation of amygdalopetal cholinergic afferents from basal forebrain. 2. Bath application of carbachol depolarized pyramidal cells with an associated increase in input resistance (Ri), reduced the slow after-hyperpolarization (AHP) that followed a series of current-evoked action potentials and blocked spike frequency accommodation. All of these effects were reversed by the muscarinic antagonist atropine but not by the nicotinic antagonist hexamethonium. 3. Electrical stimulation of amygdaloid afferents within the external capsule evoked a series of synaptic potentials consisting of a non-cholinergic fast excitatory postsynaptic potential (EPSP), followed by early and late inhibitory postsynaptic potentials (IPSPs). Each of these synaptic potentials was reduced by carbachol in an atropine-sensitive manner. 4. Local application of carbachol to pyramidal cells produced a short-latency hyperpolarization followed by a prolonged depolarization. The hyperpolarization and depolarization to carbachol were blocked by atropine but not hexamethonium. 5. The carbachol-induced hyperpolarization was associated with a decrease in Ri and had a reversal potential nearly identical to that of the early IPSP. The inhibitory response was blocked by perfusion of medium containing tetrodotoxin (TTX), bicuculline or picrotoxin, while the subsequent depolarization was unaffected. On the basis of these data, it is concluded that the muscarinic hyperpolarization is mediated through the rapid excitation of presynaptic GABAergic interneurons in the slice. 6. The findings that the carbachol-induced depolarization was associated with an increase in Ri, often had a reversal potential below -80 mV, was sensitive to changes in extracellular potassium concentration and was blocked by intracellular ionophoresis of the potassium channel blocker caesium suggest that it resulted from a muscarinic blockade of one or more potassium conductances. 7. Repetitive stimulation of sites within the slice containing cholinergic afferents evoked a series of fast EPSPs followed by IPSPs. These non-cholinergic potentials were followed by a slow EPSP that lasted from 10 s-4 min. The slow EPSP was enhanced by eserine and blocked by atropine. It was also blocked by TTX or cadmium, indicating that it was dependent on spike propagation and calcium-dependent release of acetylcholine (ACh). 8. Stimulation of cholinergic afferents in the slice mimicked other effects produced by carbachol including blockade of the slow AHP and accommodation of action potential discharge and these actions were potentiated by eserine and blocked by atropine.(ABSTRACT TRUNCATED AT 400 WORDS)