Dopaminergic modulation of sodium current in hippocampal neurons via cAMP-dependent phosphorylation of specific sites in the sodium channel alpha subunit.

Phosphorylation of brain Na+ channel alpha subunits by cAMP-dependent protein kinase (PKA) decreases peak Na+ current in cultured brain neurons and in mammalian cells and Xenopus oocytes expressing cloned brain Na+ channels. We have studied PKA regulation of Na+ channel function by activation of D1-like dopamine receptors in acutely isolated hippocampal neurons using whole-cell voltage-clamp recording techniques. The D1 agonist SKF 81297 reversibly reduced peak Na+ current in a concentration-dependent manner.

Phosphorylation at a single site in the rat brain sodium channel is necessary and sufficient for current reduction by protein kinase A.

Voltage-gated sodium channels respond to excitatory inputs in nerve cells, generating spikes of depolarization at axon hillock regions and propagating the initial rising phase of action potentials through axons. It previously has been shown that protein kinase A (PKA) attenuates sodium current amplitude 20-50% by phosphorylating serines located in the I-II linker of the sodium channel. We have tested the individual contributions of five PKA consensus sites in the I-II linker by measuring sodium currents expressed in Xenopus oocytes during conditions of PKA induction.

Topology of the Shaker potassium channel probed with hydrophilic epitope insertions.

The structure of the Shaker potassium channel has been modeled as passing through the cellular membrane eight times with both the NH2 and COOH termini on the cytoplasmic side (Durrell, S.R., and H.

Frequency selective bolometers.

We propose a concept for radiometry in the millimeter, the submillimeter, and the far-IR spectral regions, the frequency selective bolometer (FSB). This system uses a bolometer as a coupled element of a tuned quasi-optical interference filter in which the absorption, the transmission, and the reflection characteristics of the filter depend on the frequency in a controlled manner. Several FSB's can be cascaded within a straight light pipe to produce a high-efficiency, compact, multiband radiometer.

A missense mutation in the sodium channel Scn8a is responsible for cerebellar ataxia in the mouse mutant jolting.

The voltage-gated sodium channel Scn8a is broadly distributed in brain and spinal cord. We have identified a missense mutation in Scn8a that is associated with cerebellar ataxia in the jolting mutant, a mild allele of the "motor endplate disease" locus. The jolting mutation results in substitution of Thr for an evolutionarily conserved Ala residue in the cytoplasmic S4-S5 linker of domain III.

Phosphorylation of brain sodium channels in the I--II linker modulates channel function in Xenopus oocytes.

Voltage-gated sodium channels, which initiate action potentials in mammalian brain neurons, are modulated functionally by cAMP-dependent protein kinase A (PKA), resulting in reduced sodium current amplitude. Comparing brain and muscle sodium channels, we show that only the brain channel is modulated by PKA. The brain sodium channel I-II linker is both necessary and sufficient for PKA modulation, as shown by exchanging the I-II linker regions of the two channels.

The adult rat brain beta 1 subunit modifies activation and inactivation gating of multiple sodium channel alpha subunits.

The sodium channel from adult rat brain consists of a high molecular weight alpha subunit associated with low molecular weight subunits termed beta 1 and beta 2. Coexpression of beta 1 accelerates the macroscopic kinetics of inactivation of adult rat brain IIA, embryonic rat brain III, and rat skeletal muscle SkM1 sodium channel alpha subunits. In addition, beta 1 accelerates the kinetics of activation, as observed with a non-inactivating rat brain IIA mutant.

Point mutations in IIS4 alter activation and inactivation of rat brain IIA Na channels in Xenopus oocyte macropatches.

Macroscopic currents of wild-type rat brain IIA (RBIIA) and mutant Na channels were recorded in excised patches from Xenopus oocytes. A charge deletion (K859Q) and an adjacent conservative mutation (L860F) in the second domain S4 membrane-spanning region differentially altered voltage sensitivity and kinetics. Analysis of voltage dependence was confined to Na currents with fast inactivation kinetics, although RBIIA and K859Q (but not L860F) also showed proportional shifts between at least two gating modes, rendering currents with fast or slow inactivation kinetics, respectively.

A peptide segment critical for sodium channel inactivation functions as an inactivation gate in a potassium channel.

The short cytoplasmic peptide segment connecting domains III and IV of voltage-gated sodium channels (III-IV linker) is essential for fast inactivation. To test the functional similarity between the III-IV linker and the potassium channel inactivation particle, we attached the III-IV linker to the amino terminus of a noninactivating potassium channel. This chimeric channel inactivated rapidly and displayed biophysical properties similar to Shaker A-type potassium channels.

Diagnosis of Giardia duodenalis infection in Bangladeshi infants: faecal antigen capture ELISA.

An enzyme-linked immunosorbent assay (ELISA) to detect antigens of Giardia duodenalis in faeces was evaluated as a diagnostic tool by testing faecal samples collected during a cohort study of 229 infants living in an urban slum in Dhaka, Bangladesh. Faecal samples had been collected at enrollment, on a routine monthly basis, and repeatedly during episodes of diarrhoea and infection with Giardia, and a portion of all samples was frozen in saline. A direct smear of all had been examined by microscopy and again after concentrating cysts by ether sedimentation.

Accessory subunits and sodium channel inactivation.

Recent studies have shown that the accessory subunits of the voltage-gated sodium channel can modify its inactivation properties. Other studies have demonstrated that the cytoplasmic linker between domains III and IV is critical for fast inactivation. Future work should help to define the mechanisms by which these processes occur, and how mutations affecting sodium channel inactivation result in human neurological diseases.

Site-directed mutagenesis of the putative pore region of the rat IIA sodium channel.

We have used site-directed mutagenesis to examine the functional role of each of the eight acidic amino acid residues in the region between proposed transmembrane segments 5 and 6 (S5-S6) of domain II of the rat brain IIA sodium channel alpha subunit. The mutant sodium channels were expressed in Xenopus oocytes and analyzed by two-microelectrode voltage clamping with respect to voltage-dependent activation, inactivation, ion selectivity, and sensitivity to the pore-blocking neurotoxins tetrodotoxin (TTX) and saxitoxin (STX). None of the mutations had significant effects on voltage-dependent gating, ion selectivity, or block by protons or calcium.

A cluster of hydrophobic amino acid residues required for fast Na(+)-channel inactivation.

The inward Na+ current underlying the action potential in nerve is terminated by inactivation. The preceding report shows that deletions within the intracellular linker between domains III and IV remove inactivation, but mutation of conserved basic and paired acidic amino acids has little effect. Here we show that substitution of glutamine for three clustered hydrophobic amino acids, Ile-1488, Phe-1489, and Met-1490, completely removes fast inactivation.

Amino acid residues required for fast Na(+)-channel inactivation: charge neutralizations and deletions in the III-IV linker.

The cytoplasmic linker connecting domains III and IV of the voltage-gated Na+ channel is thought to be involved in fast inactivation. This linker is highly conserved among the various Na+ channels that have been cloned. In the rat brain IIA Na+ channel, it consists of 53 amino acids of which 15 are charged.

Protein kinase A phosphorylation enhances sodium channel currents in Xenopus oocytes.

The voltage-sensitive rat brain sodium channel is known to be phosphorylated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA), but the functional significance of that phosphorylation is unknown. We have shown that rat brain sodium channel currents expressed in Xenopus oocytes were enhanced by induction of PKA activity. Stimulation of the beta 2-adrenergic receptor or treatment with dibutyryl cAMP resulted in increased sodium current amplitudes without affecting the voltage dependence of channel activation or inactivation.

Primary structure and functional expression of the beta 1 subunit of the rat brain sodium channel.

Voltage-sensitive sodium channels are responsible for the initiation and propagation of the action potential and therefore are important for neuronal excitability. Complementary DNA clones encoding the beta 1 subunit of the rat brain sodium channel were isolated by a combination of polymerase chain reaction and library screening techniques. The deduced primary structure indicates that the beta 1 subunit is a 22,851-dalton protein that contains a single putative transmembrane domain and four potential extracellular N-linked glycosylation sites, consistent with biochemical data.

A voltage-dependent gating transition induces use-dependent block by tetrodotoxin of rat IIA sodium channels expressed in Xenopus oocytes.

We have utilized molecular biological techniques to demonstrate that rat IIA sodium channels expressed in Xenopus oocytes were blocked by tetrodotoxin (TTX) in a use-dependent manner. This use dependence was the result of an increased affinity of the channels for TTX upon depolarization, most likely due to a conformational change in the channel. Using a mutant with a slower macroscopic rate of inactivation, we have demonstrated that this conformational change is not the transition into the fast-inactivated state.

Expression and chromosomal localization of a lymphocyte K+ channel gene.

We recently isolated a family of three closely related mouse K+ channel genes (MK1, MK2, and MK3) with coding regions contained in single uninterrupted exons. Here we have used patch-clamp recordings from Xenopus oocytes injected with mRNA to show that MK3 encodes a channel with biophysical and pharmacological properties indistinguishable from those of voltage-gated type n K+ channels in T cells. In addition, we used the polymerase chain reaction to demonstrate the presence of MK3 mRNA in T cells.

Inactivation of cloned Na channels expressed in Xenopus oocytes.

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J.