Subunit Composition Of Channels Research Articles

Antisense oligonucleotides induce functional deletion of ligand gated ion channels in cultured neurons and brain explants

The in situ application of the antisense technique for the study of ligand gated channels is discussed here. Using antisense oligodeoxynucleotides to downregulate a gene of interest means being confronted with a number of choices that will determine the success. These include choosing a target sequence, considering chemical modifications of the oligo as well as its length and estimation of the turnover of the target protein in order to set up the treatment schedule. In this paper a short overview of technical aspects of the antisense approach on primary cultured neurons and brain slice cultures is presented. In addition, the effects of antisense oligos on the expression of neuronal nicotinic acetylcholine receptors and GABA A receptors are discussed: Patch-clamp recordings of neurons treated with specific antisense oligos targeted at individual subunits showed a clear downregulation of the expression of native ligand gated channels. Moreover, in a number of experiments novel channel types with altered properties were observed following antisense treatment. Thus, non-targeted channel subunits that remain expressed after antisense deletion, may aggregate to form novel channel types that are normally not present. Alternatively, the translational arrest of a protein may be accompanied by compensatory changes in the synthesis and/or targeting of other channel subunits to the cell surface. The antisense technique enables identification of the functional contribution of individual channel subunits to endogenous channel activity in the central nervous system. As such it paves the way to the elucidation of in vivo channel-subunit composition and channel functions, of post- as well as pre-synaptic ligand gated channel receptors.

Voltage-gated Potassium Channel Inhibitors

Forty years have transpired since tetraethylammonium was first used to selectively inhibit the potassiuin conductance in squid axons. Since then, a large body of work has emerged describing inhibitors of voltage-gated potassium currents in a variety of cells. The advent of molecular cloning techniques and the cloning of the potassium channel encoded by the Shaker locus in Drosophila has enabled detailed structure­ function studies of several potassium channel subunits. These breakthroughs have also recently enabled studies of the "toxinology" and pharmacology of specific potassium channel subunits expressed heterologously in Xenopus oocytes and other cells. Here we describe the results of some of those efforts, focusing in particular on our work with four members of the Shaker subfamily of potassium channel a-subunits: Kvl.1 through Kvl.4. These subunits are expressed in the central nervous system and other tissues of rodents, and are highly homologous to corresponding subunits expressed in humans. We provide a profile of potency and selectivity for.five snake dendrotoxins as well as several scorpion toxins for these potassium channel subunits expressed in Xenopus oocytes. We also provide similar data for four other peptide toxins and several nonpeptide compounds that had previously been shown to inhibit potassium currents. We discuss several potential clinical applications of potassium channel inhibitors, including demyelinating diseases such as multiple sclerosis, immunosuppression, cardiac arrhythmias, neurodegenerative and psychiatric diseases. Further progress will require, among other things, a greater understanding of the expression patterns of potassium channel subunits in the CNS and elsewhere as well as knowledge of the specific subunit composition of heteromultimeric channels.

Porcine aortic endothelial gap junctions: identification and permeation by caged InsP3.

Gap junction channels permit the direct intercellular transfer of ions and small molecules and allow electrotonic coupling within tissues. Porcine aortic endothelial cells were extensively coupled, as assessed by gap junctional transfer of Lucifer yellow and the fluorescent calcium indicators fluo-3 and furaptra, but were not permeable to rhodamine B isothiocyanate-dextran 10S. The subunit composition of gap junction channels of porcine aortic endothelial cells was characterised using both northern blot analysis and RT-PCR techniques. Messenger RNA encoding connexins 37 and 43, but not 26, 32 or 40, were found in freshly isolated and cultured porcine aortic endothelial cells. Western blots using antipeptide antibodies raised to unique sequences of connexins 37, 40 and 43 showed the presence of connexins 37 and 43, but no connexin 40 was detected. Immunostaining with anticonnexin 43 antibodies showed extensive punctate fluorescent decoration of contacting membranes, whilst antibodies to connexin 37 showed predominantly intracellular staining. Caged InsP3 was found to readily permeate endothelial gap junctions. These results show that primary cultures of porcine aortic endothelial cells express connexin 37 and 43, and provide strong evidence that the second messenger molecule InsP3 can permeate porcine endothelial gap junctions.

Altered K+ channel subunit composition following hormone induction of Kv1.5 gene expression.

Diverse voltage-gated K+ channels are produced by tetramerization of channel alpha subunits that can be encoded by multiple genes. In GH3 pituitary cells, the Kv1.4 and Kv1.5 subunit genes are constitutively expressed, and dexamethasone selectively up-regulates expression of the latter gene. Here we use subunit-specific antibodies to determine the effect of Kv1.5 gene induction on expression of homomeric and heteromeric channels. In control cells, almost all detectable Kv1.4 proteins are associated with Kv1.5 protein, whereas only approximately 30% Kv1.5 protein is present as Kv1.4/Kv1.5 heteromeric channels. Dexamethasone treatment doubled expression of Kv1.5 homomeric channels but only modestly up-regulated Kv1.4/Kv1.5 heteromeric complexes. The steroid treatment similarly increased total cellular and cell surface Kv1.5 protein without affecting Kv1.4 proteins in either fraction. Thus, hormone induction of one K+ channel subunit gene differentially influences expression of cell surface homomeric and heteromeric channels leading to specific alteration of excitability.

Effects of Ethanol on Ion Channels

Ion channels play critical roles in nervous system function, from initiating rapid synaptic activity to propagation of action potentials. Studies have indicated that many of the effects of ethanol on the nervous system are likely caused by the actions of ethanol on ion channels. Ion channels are multimeric structures that gate ions through subtle changes in tertiary structure. Ethanol readily enters molecular sites within multimeric ion channels, modifying intermolecular forces and bonds that are important for the open-close-inactivation kinetic properties of channels. The diversity of channel composition caused by the multimeric structure results in subtypes of channels that have a spectrum of sensitivity to ethanol that translates into brain regional differences in ethanol sensitivity, in part caused by differences in ion channel subunit composition. Ethanol has been shown to affect both receptor-activated ion channels and voltage-gated ion channels. The acute intoxicating and incoordinating effects of ethanol are probably related to inhibition of subtypes of NMDA-glutamate receptor ion channels and potentiation of certain subtypes of GABAA receptor ion channels. Effects on these channels, as well as glycine, nicotinic cholinergic, serotonergic, and other ion channels, likely contribute to the euphoric, sedative, and other acute actions of ethanol. Changes in ion channel subunit composition, density, and properties probably also contribute to ethanol tolerance, dependence, withdrawal hyperexcitability, and neurotoxicity. A substantial number of studies have implicated glutamate NMDA receptor, GABAA, and L-type voltage-gated calcium channels in the adaptive changes in the brain during chronic ethanol exposure. The diversity of ion channels subunits, their prominent role in brain function, and ethanol action are likely to make them important contributors to alcoholism and alcohol abuse.

Subunit composition of minK potassium channels.

Expression of minK protein in Xenopus oocytes induces a slowly activating, voltage-dependent, potassium-selective current. Point mutations in minK that alter current gating kinetics, ion selectivity, pharmacology, and response to protein kinase C all support the notion that minK is a structural protein for a channel-type transporter. Yet, minK has just 130 amino acids and a single transmembrane domain. Though larger cloned potassium channels form functional channels through tetrameric subunit association, the subunit composition of minK is unknown. Subunit stoichiometry was determined by coexpression of wild-type minK and a dominant lethal point mutant of minK, which reaches the plasma membrane but passes no current. The results support a model for complete minK potassium channels in which just two minK monomers are present, with other, as yet unidentified, non-minK subunits.

Patch-clamp recordings reveal powerful GABAergic inhibition in dentate hilar neurons

Whole-cell and cell-attached patch-clamp recordings were used to investigate the nature of GABAA receptor-mediated inhibition in the adult rat dentate gyrus in standard 400-microns-thick horizontal slices. In the presence of the glutamate receptor antagonists D-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, whole-cell voltage-clamp experiments with chloride-filled electrodes ([Cl-]in = [Cl-]out) revealed a high degree of spontaneous activity (10-60 Hz) in all hilar neurons (HNs) recorded with access resistances lower than 20 M omega. The events were inward at negative holding potentials, reversed at around the Cl- equilibrium potential, and were completely abolished by the specific antagonists of the GABAA receptor channel picrotoxin and SR-95531 in a reversible manner, indicating that they were spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by GABAA receptors. The majority of the sIPSCs were TTX-insensitive miniature currents resulting from the action potential-independent release of GABA. The 10-90% rise times and the monoexponential decay time constants of the sIPSCs were significantly longer in HNs than those found in neighboring granule cells (GCs). Furthermore, the decay time constant of the hilar sIPSCs was not voltage dependent, contrary to the voltage dependency of the decay time constant of the sIPSCs recorded from GCs. As HNs have longer electrotonic length than GCs do, dendritic filtering may contribute to the kinetic differences. Non-stationary fluctuation analysis showed that whereas the number of channels open at the peak of individual sIPSCs was similar, the single-channel conductances significantly differed between the two cell groups. The 21% smaller single-channel conductance and the existence of electrotonically close GABAergic synapses on HNs indicate that dendritic filtering alone cannot explain the differences between HNs and GCs. The distinct subunit composition of the GABAA receptor channels in HNs and GCs may also be responsible for the altered kinetics of IPSCs in HNs. However, the subunit specific benzodiazepine agonist zolpidem (3 microns) prolonged the monoexponential decay time constants in both HNs and GCs. Thus, differences between the GABAA receptors of the two cell types are not due to a simple all-or-none presence/absence of the alpha 5 subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium channel beta subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain.

Complementary DNAs encoding three novel and distinct beta subunits (CaB2a, CaB2b and CaB3) of the high voltage activated (L-type) calcium channel have been isolated from rabbit heart. Their deduced amino acid sequence is homologous to the beta subunit originally cloned from skeletal muscle (CaB1). CaB2a and CaB2b are splicing products of a common primary transcript (CaB2). Northern analysis and specific amplification of CaB2 and CaB3 specific cDNAs by polymerase chain reactions showed that CaB2 is predominantly expressed in heart, aorta and brain, whereas CaB3 is most abundant in brain but also present in aorta, trachea, lung, heart and skeletal muscle. A partial DNA sequence complementary to a third variant of the CaB2 gene, subtype CaB2c, has also been cloned from rabbit brain. Coexpression of CaB2a, CaB2b and CaB3 with alpha 1heart enhances not only the expression in the oocyte of the channel directed by the cardiac alpha 1 subunit alone, but also effects its macroscopic characteristics such as drug sensitivity and kinetics. These results together with the known alpha 1 subunit heterogeneity, suggest that different types of calcium currents may depend on channel subunit composition.

Subunit composition of skeletal muscle transverse tubule calcium channels evaluated with the 1,4-dihydropyridine photoaffinity probe, [3H]azidopine.

The arylazide 1,4-dihydropyridine, [3H]azidopine, binds with high affinity to calcium channels in partially purified guinea-pig skeletal muscle transverse tubule membranes. Upon brief exposure to u.v. light, [3H]azidopine incorporates covalently into transverse tubule membrane proteins, as judged by SDS-PAGE. After alkylation of sulfhydryl groups with N-ethylmaleimide three specifically labelled bands of mol wts. 240 kd, 158 kd and 99 kd are always observed with fluorography after one-dimensional SDS-PAGE. Two other specific bands with mol. wts. of 52 kd and 55 kd, respectively, were sometimes observed. Two-dimensional SDS-PAGE (non-reduced but alkylated in the first dimension and reduced in the second dimension) revealed that the 240-kd band after reduction migrates with a mol. wt. of 99 kd. The 158-kd and 99-kd bands do not change in mobility. It is suggested that [3H]azidopine binds in such a way that the arylazide moiety of the ligand comes into contact with at least three calcium channel components: the A component of mol. wt. 240 kd, the B component of mol. wt. 158 kd and a C component of mol. wt. 99 kd. B and C are non-covalently bonded subunits of the channel, whereas A could be a heterodimer consisting of B and C, linked by disulfide bonds. Subunits of smaller mol. wt. may be also part of the ionic pore. Photolabelling of transverse tubule membranes after high energy irradiation with 10 MeV electrons supports this interpretation.