Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.

Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.

NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels.

NO causes pulmonary vasodilation in patients with pulmonary hypertension. In pulmonary arterial smooth muscle cells, the activity of voltage-gated K+ (Kv) channels controls resting membrane potential. In turn, membrane potential is an important regulator of the intracellular free calcium concentration ([Ca2+]i) and pulmonary vascular tone. We used patch clamp methods to determine whether the NO-induced pulmonary vasodilation is mediated by activation of Kv channels. Quantitative fluorescence microscopy was employed to test the effect of NO on the depolarization-induced rise in [Ca2+]i. Blockade of Kv channels by 4-aminopyridine (5 mM) depolarized pulmonary artery myocytes to threshold for initiation of Ca2+ action potentials, and thereby increased [Ca2+]i. NO (approximately 3 microM) and the NO-generating compound sodium nitroprusside (5-10 microM) opened Kv channels in rat pulmonary artery smooth muscle cells. The enhanced K+ currents then hyperpolarized the cells, and blocked Ca(2+)-dependent action potentials, thereby preventing the evoked increases in [Ca2+]i. Nitroprusside also increased the probability of Kv channel opening in excised, outside-out membrane patches. This raises the possibility that NO may act either directly on the channel protein or on a closely associated molecule rather than via soluble guanylate cyclase. In isolated pulmonary arteries, 4-aminopyridine significantly inhibited NO-induced relaxation. We conclude that NO promotes the opening of Kv channels in pulmonary arterial smooth muscle cells. The resulting membrane hyperpolarization, which lowers [Ca2+]i, is apparently one of the mechanisms by which NO induces pulmonary vasodilation.

A biologic function for an "orphan" messenger: D-myo-inositol 3,4,5,6-tetrakisphosphate selectively blocks epithelial calcium-activated chloride channels.

Inositol phosphates are a family of water-soluble intracellular signaling molecules derived from membrane inositol phospholipids. They undergo a variety of complex interconversion pathways, and their levels are dynamically regulated within the cytosol in response to a variety of agonists. Relatively little is known about the biological function of most members of this family, with the exception of inositol 1,4,5-trisphosphate. Specifically, the biological functions of inositol tetrakisphosphates are largely obscure. In this paper, we report that D-myo-inositol 3,4,5,6-tetrakisphosphate (D-Ins(3,4,5,6)P4) has a direct biphasic (activation/inhibition) effect on an epithelial Ca(2+)-activated chloride channel. The effect of D-Ins(3,4,5,6)P4 is not mimicked by other inositol tetrakisphosphate isomers, is dependent on the prevailing calcium concentration, and is influenced when channels are phosphorylated by calmodulin kinase II. The predominant effect of D-Ins(3,4,5,6)P4 on phosphorylated channels is inhibitory at levels of intracellular calcium observed in stimulated cells. Our findings indicate the biological function of a molecule hitherto considered as an "orphan" messenger. They suggest that the molecular target for D-Ins(3,4,5,6)P4 is a plasma membrane Ca(2+)-activated chloride channel. Regulation of this channel by D-Ins(3,4,5,6)P4 and Ca2+ may have therapeutic implications for the disease states of both diabetic nephropathy and cystic fibrosis.

Elevation of intracellular calcium by muscarinic receptor activation induces a block of voltage-activated rat ether-à-go-go channels in a stably transfected cell line.

We have studied the properties of r-eag voltage-activated potassium channels in a stably transfected human embryonic kidney cell line. It was found that r-eag channels are rapidly and reversibly inhibited by a rise in intracellular calcium from 30 to 300 nM. The inhibition does not appear to depend on the activity of calcium-dependent kinases and phosphatases. The effect of calcium on r-eag channel activity was studied in inside-out membrane patches. Calcium inhibited r-eag channel activity with a mean IC50 of 67 nM. Activation of muscarinic receptors, generating calcium oscillations in the transfected cells, induced a synchronous inhibition of r-eag mediated outward currents. This shows that calcium can mediate r-eag current inhibition following muscarinic receptor activation. The data indicate that r-eag channels are calcium-inhibitable voltage-activated potassium channels.

A mutation that alters magnesium block of N-methyl-D-aspartate receptor channels.

N-Methyl-D-aspartate (NMDA) receptors are blocked at hyperpolarizing potentials by extracellular Mg ions. Here we present a detailed kinetic analysis of the Mg block in recombinant wild-type and mutant NMDA receptors. We find that the Mg binding site is the same in the wild-type and native hippocampal NMDA receptor channels. In the mutant channels, however, Mg ions bind with a 10-fold lower affinity. On the basis of these results, we propose that the energy well at the Mg binding site in the mutants is shallow and the binding is unstable because of an increase in the rate of dissociation. We postulate that the dipole formed by the amide group of asparagine 614 of the epsilon 1 subunit contributes to the structure of the binding site but predict that additional ligands will be involved in coordinating Mg ions.

Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage-gated Na+ channels.

Voltage-gated Na+ channels are the molecular targets of local anesthetics, class I antiarrhythmic drugs, and some anticonvulsants. These chemically diverse drugs inhibit Na+ channels with complex voltage- and frequency-dependent properties that reflect preferential drug binding to open and inactivated channel states. The site-directed mutations F1764A and Y1771A in transmembrane segment IVS6 of type IIA Na+ channel alpha subunits dramatically reduce the affinity of inactivated channels for the local anesthetic etidocaine. In this study, we show that these mutations also greatly reduce the sensitivity of Na+ channels to state-dependent block by the class Ib antiarrhythmic drug lidocaine and the anticonvulsant phenytoin and, to a lesser extent, reduce the sensitivity to block by the class Ia and Ic antiarrhythmic drugs quinidine and flecainide. For lidocaine and phenytoin, which bind preferentially to inactivated Na+ channels, the mutation F1764A reduced the affinity for binding to the inactivated state 24.5-fold and 8.3-fold, respectively, while Y1771A had smaller effects. For quinidine and flecainide, which bind preferentially to the open Na+ channels, the mutations F1764A and Y1771A reduced the affinity for binding to the open state 2- to 3-fold. Thus, F1764 and Y1771 are common molecular determinants of state-dependent binding of diverse drugs including lidocaine, phenytoin, flecainide, and quinidine, suggesting that these drugs interact with a common receptor site. However, the different magnitude of the effects of these mutations on binding of the individual drugs indicates that they interact in an overlapping, but nonidentical, manner with a common receptor site. These results further define the contributions of F1764 and Y1771 to a complex drug receptor site in the pore of Na+ channels.

Probing the pore region of recombinant N-methyl-D-aspartate channels using external and internal magnesium block.

Mg2+ ions block N-methyl-D-aspartate (NMDA) channels by entering the pore from either the extracellular or the cytoplasmic side of the membrane in a voltage-dependent manner. We have used these two different block phenomena to probe the structure of the subunits forming NMDA channels. We have made several amino acid substitutions downstream of the Q/R/N site in the TMII region of both NR1 and NR2A subunits. Mutant NR1 subunits were coexpressed with wild-type NR2A subunits and vice versa in Xenopus oocytes. We found that individually mutating the first two amino acid residues downstream to the Q/R/N site affects mostly the block by external Mg2+. Mutations of residues five to seven positions downstream of the Q/R/N site do not influence the external Mg2+ block, but clearly influence the block by internal Mg2+. These data add support to the hypothesis that there are two separate binding sites for external and internal Mg2+ block. They also indicate that the C-terminal end of TMII contributes to the inner vestibule of the pore of NMDA channels and thus provide additional evidence that TMII forms a loop that reemerges toward the cytoplasmic side of the membrane.

Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry.

Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.

Isoform-specific interaction of the alpha1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP-25.

Presynaptic Ca2+ channels 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. Here we report isoform-specific, stoichiometric interaction of the BI and rbA isoforms of the alpha1A subunit of P/Q-type Ca2+ channels with the presynaptic membrane proteins syntaxin and SNAP-25 in vitro and in rat brain membranes. The BI isoform binds to both proteins, while only interaction with SNAP-25 can be detected in vitro for the rbA isoform. The synaptic protein interaction ("synprint") site involves two adjacent segments of the intracellular loop connecting domains II and III between amino acid residues 722 and 1036 of the BI sequence. This interaction is competitively blocked by the corresponding region of the N-type Ca2+ channel, indicating that these two channels bind to overlapping regions of syntaxin and SNAP-25. Our results provide a molecular basis for a physical link between Ca2+ influx into nerve terminals and subsequent exocytosis of neurotransmitters at synapses that have presynaptic Ca2+ channels containing alpha1A subunits.

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of micro1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.

Receptor stimulation causes slow inhibition of IRK1 inwardly rectifying K+ channels by direct protein kinase A-mediated phosphorylation.

Strongly rectifying IRK-type inwardly rectifying K+ channels are involved in the control of neuronal excitability in the mammalian brain. Whole-cell patch-clamp experiments show that cloned rat IRK1 (Kir 2.1) channels, when heterologously expressed in mammalian COS-7 cells, are inhibited following the activation of coexpressed serotonin (5-hydroxytryptamine) type 1A receptors by receptor agonists. Inhibition is mimicked by internal perfusion with GTP[gamma-S] and elevation of internal cAMP concentrations. Addition of the catalytic subunits of protein kinase A (PKA) to the internal recording solution causes complete inhibition of wild-type IRK1 channels, but not of mutant IRK1(S425N) channels in which a C-terminal PKA phosphorylation site has been removed. Our data suggest that in the nervous system serotonin may negatively control IRK1 channel activity by direct PKA-mediated phosphorylation.

Spatial localization of calcium channels in giant fiber lobe neurons of the squid (Loligo opalescens).

Whole-cell voltage clamp was used to investigate the properties and spatial distribution of fast-deactivating (FD) Ca channels in squid giant fiber lobe (GFL) neurons. Squid FD Ca channels are reversibly blocked by the spider toxin omega-Agatoxin IVA with an IC50 of 240-420 nM with no effect on the kinetics of Ca channel gating. Channels with very similar properties are expressed in both somatic and axonal domains of cultured GFL neurons, but FD Ca channel conductance density is higher in axonal bulbs than in cell bodies at all times in culture. Channels presumably synthesized during culture are preferentially expressed in the growing bulbs, but bulbar Ca conductance density remains constant while Na conductance density increases, suggesting that processes determining the densities of Ca and Na channels in this extrasomatic domain are largely independent. These observations suggest that growing axonal bulbs in cultured GFL neurons are not composed entirely of "axonal" membranes because FD Ca channels are absent from the giant axon in situ but, rather, suggest a potential role for FD Ca channels in mediating neurotransmitter release at the motor terminals of the giant axon.

N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.

Evidence that free polyunsaturated fatty acids modify Na+ channels by directly binding to the channel proteins.

The effects of free polyunsaturated fatty acids (PUFA) on the binding of ligands to receptors on voltage-sensitive Na+ channels of neonatal rat cardiac myocytes were assessed. The radioligand was [benzoyl-2,5-(3)H] batrachotoxinin A 20alpha-benzoate ([(3)H]BTXB), a toxin that binds to the Na+ channel. The PUFA that have been shown to be antiarrhythmic, including eicosapentaenoic acid (EPA; C20:5n-3), docosahexaenoic acid (DHA; C22:6n-3), eicosatetraynoic acid (ETYA), linolenic acid (C18:3n-3), and linoleic acid (C18:2n-6), inhibited [(3)H]BTXB binding in a dose-dependent fashion with IC50 values of 28-35 microM, whereas those fatty acids that have no antiarrhythmic effects including saturated fatty acid (stearic acid, C18:0), monounsaturated fatty acid (oleic acid; C18:1n-9), and EPA methyl ester did not have a significant effect on [(3)H]BTXB binding. Enrichment of the myocyte membrane with cholesterol neither affected [(3)H]BTXB binding when compared with control cells nor altered the inhibitory effects of PUFA on [(3)H]BTXB binding. Scatchard analysis of [(3)H]BTXB binding showed that EPA reduced the maximal binding without altering the Kd for [(3)H]BTXB binding, indicating allosteric inhibition. The inhibition by EPA of [(3)H]BTXB binding was reversible (within 30 min) when delipidated bovine serum albumin was added. The binding of the PUFA to this site on the Na+ channel is reversible and structure-specific and occurs at concentrations close to those required for apparent antiarrhythmic effects and a blocking effect on the Na+ current, suggesting that binding of the PUFA at this site relates to their antiarrhythmic action.

Determinants of the G protein-dependent opioid modulation of neuronal calcium channels.

The modulation of a family of cloned neuronal calcium channels by stimulation of a coexpressed mu opioid receptor was studied by transient expression in Xenopus oocytes. Activation of the morphine receptor with the synthetic enkephalin [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) resulted in a rapid inhibition of alpha1A (by approximately 20%) and alpha1B (by approximately 55%) currents while alpha1C and alpha1E currents were not significantly affected. The opioid-induced effects on alpha1A and alpha1B currents were blocked by pertussis toxin and the GTP analogue guanosine 5'-[beta-thio]diphosphate. Similar to modulation of native calcium currents, DAMGO induced a slowing of the activation kinetics and exhibited a voltage-dependent inhibition that was partially relieved by application of strong depolarizing pulses. alpha1A currents were still inhibited in the absence of coexpressed Ca channel alpha2 and beta subunits, suggesting that the response is mediated by the alpha1 subunit. Furthermore, the sensitivity of alpha1A currents to DAMGO-induced inhibition was increased approximately 3-fold in the absence of a beta subunit. Overall, the results show that the alpha1A (P/Q type) and the alpha1B (N type) calcium channels are selectively modulated by a GTP-binding protein (G protein). The results raise the possibility of competitive interactions between beta subunit and G protein binding to the alpha1 subunit, shifting gating in opposite directions. At presynaptic terminals, the G protein-dependent inhibition may result in decreased synaptic transmission and play a key role in the analgesic effect of opioids and morphine.