Molecular properties of brain sodium channels: an important target for anticonvulsant drugs.

The voltage-gated sodium channels that are responsible for action potential generation in central neurons are important targets for the actions of antiepileptic drugs. These channels consist of a complex of three glycoprotein subunits: a pore-forming alpha subunit of 260 kd associated noncovalently with a beta 1 subunit of 36 kd and disulfide-linked to a beta 2 subunit of 33 kd. The alpha subunit forms a functional voltage-gated sodium channel by itself, whereas the beta 1 and beta 2 subunits modulate channel gating.

Dopaminergic modulation of voltage-gated Na+ current in rat hippocampal neurons requires anchoring of cAMP-dependent protein kinase.

Activation of D1-like dopamine (DA) receptors reduces peak Na(+) current in acutely isolated hippocampal neurons via a modulatory mechanism involving phosphorylation of the Na(+) channel alpha subunit by cAMP-dependent protein kinase (PKA). Peak Na(+) current is reduced 20-50% in the presence of the D1 agonist SKF 81297 or the PKA activator Sp-5,6-dichloro-l-beta-d-ribofuranosyl benzimidazole-3',5'-cyclic monophosphorothionate (cBIMPS). Co-immunoprecipitation experiments show that Na(+) channels are associated with PKA and A-kinase-anchoring protein 15 (AKAP-15), and immunocytochemical labeling reveals their co-localization in the cell bodies and proximal dendrites of hippocampal pyramidal neurons.

Interactions of presynaptic Ca2+ channels and snare proteins in neurotransmitter release.

N- and P/Q-type Ca2+ channels are localized in high density in presynaptic nerve terminals and are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. These Ca2+ channels can be purified from brain as a complex with SNARE proteins, which are involved in exocytosis.

Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons.

Activation of D1-like dopamine (DA) receptors reduces peak Na+ current in acutely isolated hippocampal neurons through phosphorylation of the alpha subunit of the Na+ channel by cAMP-dependent protein kinase (PKA). Here we report that neuromodulation of Na+ currents by DA receptors via PKA is voltage-dependent in the range of -110 to -70 mV and is also sensitive to concurrent activation of protein kinase C (PKC). Depolarization enhanced the ability of D1-like DA receptors to reduce peak Na+ currents via the PKA pathway.

Ca2+/calmodulin binds to and modulates P/Q-type calcium channels.

Neurotransmitter release at many central synapses is initiated by an influx of calcium ions through P/Q-type calcium channels, which are densely localized in nerve terminals. Because neurotransmitter release is proportional to the fourth power of calcium concentration, regulation of its entry can profoundly influence neurotransmission. N- and P/Q-type calcium channels are inhibited by G proteins, and recent evidence indicates feedback regulation of P/Q-type channels by calcium.

Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release.

Electrophysiological studies of neurons reveal different Ca2+ currents designated L-, N-, P-, Q-, R-, and T-type. High-voltage-activated neuronal Ca2+ channels are complexes of a pore-forming alpha 1 subunit of about 190-250 kDa, a transmembrane, disulfide-linked complex of alpha 2 and delta subunits, and an intracellular beta subunit, similar to the alpha 1, alpha 2 delta, and beta subunits previously described for skeletal muscle Ca2+ channels. The primary structures of these subunits have all been determined by homology cDNA cloning using the corresponding subunits of skeletal muscle Ca2+ channels as probes.

Block of brain sodium channels by peptide mimetics of the isoleucine, phenylalanine, and methionine (IFM) motif from the inactivation gate.

Inactivation of sodium channels is thought to be mediated by an inactivation gate formed by the intracellular loop connecting domains III and IV. A hydrophobic motif containing the amino acid sequence isoleucine, phenylalanine, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM motif, when applied to the cytoplasmic side of these channels, produce two types of block: fast block, which resembles the inactivation process, and slow, use-dependent block stimulated by strong depolarizing pulses.

Solution structure of the sodium channel inactivation gate.

The sodium channel initiates action potentials by opening in response to membrane depolarization. Fast channel inactivation, which is required for proper physiological function, is mediated by a cytoplasmic loop proposed to occlude the ion pore via a hinged lid mechanism with the triad IFM serving as a hydrophobic "latch". The NMR solution structure of the isolated inactivation gate reveals a stably folded core comprised of an alpha-helix capped by an N-terminal turn, supporting a model in which the tightly folded core containing the latch motif pivots on a more flexible hinge region to occlude the pore during inactivation.

Exclusion of the SCN2B gene as candidate for CMT4B.

Charcot-Marie-Tooth disease type 4B (CMT4B) is a demyelinating autosomal recessive motor and sensory neuropathy characterised by focally folded myelin sheaths in the peripheral nerve. The CMT4B gene has been localised by homozygosity mapping and haplotype sharing in the 11q23 region. A cDNA encoding for the beta 2 subunit of the human brain sodium channel, SCN2B, has been recently assigned to the same chromosomal interval by FISH.

Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses.

We studied how Ca2+ influx through different subtypes of Ca2+ channels couples to release at a calyx-type terminal in the rat medial nucleus of the trapezoid body by simultaneously measuring the presynaptic Ca2+ influx evoked by a single action potential and the EPSC. Application of subtype-specific toxins showed that Ca2+ channels of the P/Q-, N-, and R-type controlled glutamate release at a single terminal. The Ca2+ influx through the P/Q-type channels triggered release more effectively than Ca2+ influx through N- or R-type channels.

Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R.

The type IIA rat brain sodium channel is composed of three subunits: a large pore-forming alpha subunit and two smaller auxiliary subunits, beta1 and beta2. The beta subunits are single membrane-spanning glycoproteins with one Ig-like motif in their extracellular domains. The Ig motif of the beta2 subunit has close structural similarity to one of the six Ig motifs in the extracellular domain of the cell adhesion molecule contactin (also called F3 or F11), which binds to the extracellular matrix molecules tenascin-C and tenascin-R.

Evidence for a voltage-dependent enhancement of neurotransmitter release mediated via the synaptic protein interaction site of N-type Ca2+ channels.

Secretion of neurotransmitters is initiated by voltage-gated calcium influx through presynaptic, voltage-gated N-type calcium channels. These channels interact with the SNARE proteins, which are core components of the exocytosis process, via the synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their alpha1B subunit. Interruption of this interaction by competing synprint peptides inhibits fast, synchronous transmitter release.

Interaction of batrachotoxin with the local anesthetic receptor site in transmembrane segment IVS6 of the voltage-gated sodium channel.

The voltage-gated sodium channel is the site of action of more than six classes of neurotoxins and drugs that alter its function by interaction with distinct, allosterically coupled receptor sites. Batrachotoxin (BTX) is a steroidal alkaloid that binds to neurotoxin receptor site 2 and causes persistent activation. BTX binding is inhibited allosterically by local anesthetics.

Voltage sensor-trapping: enhanced activation of sodium channels by beta-scorpion toxin bound to the S3-S4 loop in domain II.

Polypeptide neurotoxins alter ion channel gating by binding to extracellular receptor sites, even though the voltage sensors are in their S4 transmembrane segments. By analysis of sodium channel chimeras, a beta-scorpion toxin is shown here to negatively shift voltage dependence of activation and enhance closed state inactivation by binding to a receptor site that requires glycine 845 (Gly-845) in the S3-S4 loop at the extracellular end of the S4 segment in domain II of the alpha subunit. Toxin action requires prior depolarization to drive the S4 voltage sensors outward, but these effects are lost in the mutant G845N.

G-protein beta-subunit specificity in the fast membrane-delimited inhibition of Ca2+ channels.

We investigated which subtypes of G-protein beta subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein beta subunits. Gbeta1 and Gbeta2 strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors.

Physical link and functional coupling of presynaptic calcium channels and the synaptic vesicle docking/fusion machinery.

N- and P/Q-type calcium channels are localized in high density in presynaptic nerve terminals and are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. As outlined in the preceding article, these calcium channels can be purified from brain as a complex with SNARE proteins which are involved in exocytosis.

AKAP15 anchors cAMP-dependent protein kinase to brain sodium channels.

The voltage-sensitive sodium channel is regulated by cAMP-dependent protein kinase (PKA) phosphorylation. Using purified preparations of rat brain sodium channels, we have shown that the alpha subunit was phosphorylated by a co-purifying protein kinase. The co-purifying kinase was stimulated by cAMP and phosphorylated PKA substrate peptides.

Localization of Ca2+ channel subtypes on rat spinal motor neurons, interneurons, and nerve terminals.

Ca2+ channels in distinct subcellular compartments of neurons mediate voltage-dependent Ca2+ influx, which integrates synaptic responses, regulates gene expression, and initiates synaptic transmission. Antibodies that specifically recognize the alpha1 subunits of class A, B, C, D, and E Ca2+ channels have been used to investigate the localization of these voltage-gated ion channels on spinal motor neurons, interneurons, and nerve terminals of the adult rat. Class A P/Q-type Ca2+ channels were present mainly in a punctate pattern in nerve terminals located along the cell bodies and dendrites of motor neurons.

Regulation of ion channels by cAMP-dependent protein kinase and A-kinase anchoring proteins.

Subcellular targeting of the cAMP-dependent protein kinase is achieved, in part, through association with A-kinase anchoring proteins (AKAPs). Recent evidence suggests that specific AKAPs direct the kinase to submembrane sites to facilitate phosphorylation and modulation of a variety of ion channels. A new membrane-anchored AKAP targets cAMP-dependent protein kinase to calcium channels and enhances their regulation in multiple cell types.

Primary structure and function of an A kinase anchoring protein associated with calcium channels.

Rapid, voltage-dependent potentiation of skeletal muscle L-type calcium channels requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A kinase anchoring protein (AKAP). Here we report the isolation, primary sequence determination, and functional characterization of AKAP15, a lipid-anchored protein of 81 amino acid residues with a single amphipathic helix that binds PKA. AKAP15 colocalizes with L-type calcium channels in transverse tubules and is associated with L-type calcium channels in transfected cells.

Upregulation of L-type Ca2+ channels in reactive astrocytes after brain injury, hypomyelination, and ischemia.

Anti-peptide antibodies that specifically recognize the alpha1 subunit of class A-D voltage-gated Ca2+ channels and a monoclonal antibody (MANC-1) to the alpha2 subunit of L-type Ca2+ channels were used to investigate the distribution of these Ca2+ channel subtypes in neurons and glia in models of brain injury, including kainic acid-induced epilepsy in the hippocampus, mechanical and thermal lesions in the forebrain, hypomyelination in white matter, and ischemia. Immunostaining of the alpha2 subunit of L-type Ca2+ channels by the MANC-1 antibody was increased in reactive astrocytes in each of these forms of brain injury. The alpha1C subunits of class C L-type Ca2+ channels were upregulated in reactive astrocytes located in the affected regions in each of these models of brain injury, although staining for the alpha1 subunits of class D L-type, class A P/Q-type, and class B N-type Ca2+ channels did not change from patterns normally observed in control animals.

Molecular determinants of Na+ channel function in the extracellular domain of the beta1 subunit.

The rat brain voltage-gated Na+ channel is composed of three glycoprotein subunits: the pore-forming alpha subunit and two auxiliary subunits, beta1 and beta2, which contain immunoglobulin (Ig)-like folds in their extracellular domains. When expressed in Xenopus oocytes, beta1 modulates the gating properties of the channel-forming type IIA alpha subunit, resulting in an acceleration of inactivation. We have used a combination of deletion, alanine-scanning, site-directed, and chimeric mutagenesis strategies to examine the importance of different structural features of the beta1 subunit in the modulation of alphaIIA function, with an emphasis on the extracellular domain.

A critical role for the S4-S5 intracellular loop in domain IV of the sodium channel alpha-subunit in fast inactivation.

Na+ channel fast inactivation is thought to involve the closure of an intracellular inactivation gate over the channel pore. Previous studies have implicated the intracellular loop connecting domains III and IV and a critical IFM motif within it as the inactivation gate, but amino acid residues at the intracellular mouth of the pore required for gate closure and binding have not been positively identified. The short intracellular loops connecting the S4 and S5 segments in each domain of the Na+ channel alpha-subunit are good candidates for this role in the Na+ channel inactivation process.

Kinetic analysis of block of open sodium channels by a peptide containing the isoleucine, phenylalanine, and methionine (IFM) motif from the inactivation gate.

We analyzed the kinetics of interaction between the peptide KIFMK, containing the isoleucine, phen-ylalanine, and methionine (IFM) motif from the inactivation gate, and the brain type IIA sodium channels with a mutation that disrupts inactivation (F1489Q). The on-rate constant was concentration dependent, consistent with a bimolecular reaction with open sodium channels, while the off rates were unaffected by changes in the KIFMK concentration. The apparent Kd was approximately 33 microM at 0 mV.