The γ-aminobutyric acid type A receptor (GABAAR)-associated protein GABARAP interacts with gephyrin but is not involved in receptor anchoring at the synapse

γ-Aminobutyric acid type A receptors (GABAARs) are ligand-gated chloride channels that exist in numerous distinct subunit combinations. At postsynaptic membrane specializations, different GABAAR isoforms colocalize with the tubulin-binding protein gephyrin. However, direct interactions of GABAAR subunits with gephyrin have not been reported. Recently, the GABAAR-associated protein GABARAP was found to bind to the γ2 subunit of GABAARs. Here we show that GABARAP interacts with gephyrin in both biochemical assays and transfected cells. Confocal analysis of neurons derived from wild-type and gephyrin-knockout mice revealed that GABARAP is highly enriched in intracellular compartments, but not at gephyrin-positive postsynaptic membrane specializations. Our data indicate that GABARAP–gephyrin interactions are not important for postsynaptic GABAAR anchoring but may be implicated in receptor sorting and/or targeting mechanisms. Consistent with this idea, a close homolog of GABARAP, p16, has been found to function as a late-acting intra-Golgi transport factor.

Pentameric assembly of a neuronal glutamate transporter

Freeze-fracture electron microscopy was used to study the structure of a human neuronal glutamate transporter (EAAT3). EAAT3 was expressed in Xenopus laevis oocytes, and its function was correlated with the total number of transporters in the plasma membrane of the same cells. Function was assayed as the maximum charge moved in response to a series of transmembrane voltage pulses. The number of transporters in the plasma membrane was determined from the density of a distinct 10-nm freeze-fracture particle, which appeared in the protoplasmic face only after EAAT3 expression. The linear correlation between EAAT3 maximum carrier-mediated charge and the total number of the 10-nm particles suggested that this particle represented functional EAAT3 in the plasma membrane. The cross-sectional area of EAAT3 in the plasma membrane (48 ± 5 nm2) predicted 35 ± 3 transmembrane α-helices in the transporter complex. This information along with secondary structure models (6–10 transmembrane α-helices) suggested an oligomeric state for EAAT3. EAAT3 particles were pentagonal in shape in which five domains could be identified. They exhibited fivefold symmetry because they appeared as equilateral pentagons and the angle at the vertices was 110°. Each domain appeared to contribute to an extracellular mass that projects ≈3 nm into the extracellular space. Projections from all five domains taper toward an axis passing through the center of the pentagon, giving the transporter complex the appearance of a penton-based pyramid. The pentameric structure of EAAT3 offers new insights into its function as both a glutamate transporter and a glutamate-gated chloride channel.

Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate

Higher plants share with animals a responsiveness to the Ca2+ mobilizing agents inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular 45Ca2+ flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca2+-releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca2+ with a K1/2 = 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca2+ pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca2+ pathway distinguishes it from the InsP3- and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol/mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca2+ release is insensitive to heparin and 8-NH2-cADPR, specific inhibitors of the InsP3- and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca2+ release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca2+ release pathway is independent of cytosolic free Ca2+ and therefore incapable of operating Ca2+-induced Ca2+ release. In contrast to the sea urchin system, the NAADP-gated Ca2+ release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca2+ mobilization pathways and Ca2+ release sites might contribute to the generation of stimulus-specific Ca2+ signals in plant cells.

A cGMP-signaling pathway in a subset of olfactory sensory neurons

It is well established that signal transduction in sensory neurons of the rat olfactory epithelium involves a cAMP-signaling pathway. However, a small number of olfactory neurons specifically express cGMP-signaling components, namely a guanylyl cyclase (GC-D) and a cGMP-stimulated phosphodiesterase (PDE2). Here, we show that this subset of olfactory neurons expressing GC-D and PDE2 does also express the subunit of a cGMP-selective cyclic nucleotide-gated (CNG) channel that has been previously identified in cone photoreceptors. Further, components of the prototypical cAMP-signaling pathway could not be detected in this subpopulation of cells. These results imply that these neurons use an alternative signaling pathway, with cGMP as the intracellular messenger, and that, in these cells, the receptor current is initiated by the opening of cGMP-gated channels.

Mutations of γ-aminobutyric acid and glycine receptors change alcohol cutoff: Evidence for an alcohol receptor?

Alcohols in the homologous series of n-alcohols increase in central nervous system depressant potency with increasing chain length until a “cutoff” is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the α subunit. We now demonstrate that these residues in the glycine α1 and the γ-aminobutyric acid ρ1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine α1 receptor, whereas mutation of Ile-307 and/or Trp-328 in the γ-aminobutyric acid ρ1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.

Two functionally dependent acetylcholine subunits are encoded in a single Caenorhabditis elegans operon

The deg-3 gene from the nematode Caenorhabditis elegans encodes an α subunit of a nicotinic acetylcholine receptor that was first identified by a dominant allele, u662, which produced neuronal degeneration. Because deg-3 cDNAs contain the SL2 trans-spliced leader, we suggested that deg-3 was transcribed as part of a C. elegans operon. Here we show that des-2, a gene in which mutations suppress deg-3(u662), is the upstream gene in that operon. The des-2 gene also encodes an α subunit of a nicotinic acetylcholine receptor. As expected for genes whose mRNAs are formed from a single transcript, both genes have similar expression patterns. This coexpression is functionally important because (i) des-2 is needed for the deg-3(u662) degenerations in vivo; (ii) an acetylcholine-gated channel is formed in Xenopus oocytes when both subunits are expressed but not when either is expressed alone; and (iii) channel activity, albeit apparently altered from that of the wild-type channel, results from the expression of a u662-type mutant subunit but, again, only when the wild-type DES-2 subunit is present. Thus, the operon structure appears to regulate the coordinate expression of two channel subunits.

Origin of a chloroplast protein importer

During evolution, chloroplasts have relinquished the majority of their genes to the nucleus. The products of transferred genes are imported into the organelle with the help of an import machinery that is distributed across the inner and outer plastid membranes. The evolutionary origin of this machinery is puzzling because, in the putative predecessors, the cyanobacteria, the outer two membranes, the plasma membrane, and the lipopolysaccharide layer lack a functionally similar protein import system. A 75-kDa protein-conducting channel in the outer envelope of pea chloroplasts, Toc75, shares ≈22% amino acid identity to a similarly sized protein, designated SynToc75, encoded in the Synechocystis PCC6803 genome. Here we show that SynToc75 is located in the outer membrane (lipopolysaccharide layer) of Synechocystis PCC6803 and that SynToc75 forms a voltage-gated, high conductance channel with a high affinity for polyamines and peptides in reconstituted liposomes. These findings suggest that a component of the chloroplast protein import system, Toc75, was recruited from a preexisting channel-forming protein of the cyanobacterial outer membrane. Furthermore, the presence of a protein in the chloroplastic outer envelope homologous to a cyanobacterial protein provides support for the prokaryotic nature of this chloroplastic membrane.

Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus

Large conductance voltage- and Ca2+-dependent K+ (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers β subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and “in vivo” reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus.

Expression of γ-aminobutyric acid ρ1 and ρ1Δ450 as gene fusions with the green fluorescent protein

The functional characteristics and cellular localization of the γaminobutyric acid (GABA) ρ1 receptor and its nonfunctional isoform ρ1Δ450 were investigated by expressing them as gene fusions with the enhanced version of the green fluorescent protein (GFP). Oocytes injected with ρ1-GFP had receptors that gated chloride channels when activated by GABA. The functional characteristics of these receptors were the same as for those of wild-type ρ1 receptors. Fluorescence, because of the chimeric receptors expressed, was over the whole oocyte but was more intense near the cell surface and more abundant in the animal hemisphere. Similar to the wild type, ρ1Δ450-GFP did not lead to the expression of functional GABA receptors, and injected oocytes failed to generate currents even after exposure to high concentrations of GABA. Nonetheless, the fluorescence displayed by oocytes expressing ρ1Δ450-GFP was distributed similarly to that of ρ1-GFP. Mammalian cells transfected with the ρ1-GFP or ρ1Δ450-GFP constructs showed mostly intracellularly distributed fluorescence in confocal microscope images. A sparse localization of fluorescence was observed in the plasma membrane regardless of the cell line used. We conclude that ρ1Δ450 is expressed and transported close to, and perhaps incorporated into, the plasma membrane. Thus, ρ1- and ρ1Δ450-GFP fusions provide a powerful tool to visualize the traffic of GABA type C receptors.

α10: A determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells

We report the cloning and characterization of rat α10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the α10 nAChR subunit gene is most similar to the rat α9 nAChR, and both α9 and α10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with α10 cRNA alone or in pairwise combinations with either α2-α6 or β2-β4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of α9 and α10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric α9 channels, the α9α10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current–voltage relationship, and a biphasic response to changes in extracellular Ca2+ ions. The pharmacological profiles of homomeric α9 and heteromeric α9α10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both α9 and α10 subunits.

Single point mutations affect fatty acid block of human myocardial sodium channel α subunit Na+ channels

Suppression of cardiac voltage-gated Na+ currents is probably one of the important factors for the cardioprotective effects of the n-3 polyunsaturated fatty acids (PUFAs) against lethal arrhythmias. The α subunit of the human cardiac Na+ channel (hH1α) and its mutants were expressed in human embryonic kidney (HEK293t) cells. The effects of single amino acid point mutations on fatty acid-induced inhibition of the hH1α Na+ current (INa) were assessed. Eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced INa in HEK293t cells expressing the wild type, Y1767K, and F1760K of hH1α Na+ channels. The inhibition was voltage and concentration-dependent with a significant hyperpolarizing shift of the steady state of INa. In contrast, the mutant N406K was significantly less sensitive to the inhibitory effect of EPA. The values of the shift at 1, 5, and 10 μM EPA were significantly smaller for N406K than for the wild type. Coexpression of the β1 subunit and N406K further decreased the inhibitory effects of EPA on INa in HEK293t cells. In addition, EPA produced a smaller hyperpolarizing shift of the V1/2 of the steady-state inactivation in HEK293t cells coexpressing the β1 subunit and N406K. These results demonstrate that substitution of asparagine with lysine at the site of 406 in the domain-1-segment-6 region (D1-S6) significantly decreased the inhibitory effect of PUFAs on INa, and coexpression with β1 decreased this effect even more. Therefore, asparagine at the 406 site in hH1α may be important for the inhibition by the PUFAs of cardiac voltage-gated Na+ currents, which play a significant role in the antiarrhythmic actions of PUFAs.

Excitation–contraction uncoupling by a human central core disease mutation in the ryanodine receptor

Central core disease (CCD) is a human congenital myopathy characterized by fetal hypotonia and proximal muscle weakness that is linked to mutations in the gene encoding the type-1 ryanodine receptor (RyR1). CCD is thought to arise from Ca2+-induced damage stemming from mutant RyR1 proteins forming “leaky” sarcoplasmic reticulum (SR) Ca2+ release channels. A novel mutation in the C-terminal region of RyR1 (I4898T) accounts for an unusually severe and highly penetrant form of CCD in humans [Lynch, P. J., Tong, J., Lehane, M., Mallet, A., Giblin, L., Heffron, J. J., Vaughan, P., Zafra, G., MacLennan, D. H. & McCarthy, T. V. (1999) Proc. Natl. Acad. Sci. USA 96, 4164–4169]. We expressed in skeletal myotubes derived from RyR1-knockout (dyspedic) mice the analogous mutation engineered into a rabbit RyR1 cDNA (I4897T). Here we show that homozygous expression of I4897T in dyspedic myotubes results in a complete uncoupling of sarcolemmal excitation from voltage-gated SR Ca2+ release without significantly altering resting cytosolic Ca2+ levels, SR Ca2+ content, or RyR1-mediated enhancement of dihydropyridine receptor (DHPR) channel activity. Coexpression of both I4897T and wild-type RyR1 resulted in a 60% reduction in voltage-gated SR Ca2+ release, again without altering resting cytosolic Ca2+ levels, SR Ca2+ content, or DHPR channel activity. These findings indicate that muscle weakness suffered by individuals possessing the I4898T mutation involves a functional uncoupling of sarcolemmal excitation from SR Ca2+ release, rather than the expression of overactive or leaky SR Ca2+ release channels.

A Calcium-Selective Channel from Root-Tip Endomembranes of Garden Cress1

A Ca2+ channel from root-tip endomembranes of garden cress (Lepidium sativum L.) (LCC1) was characterized using the planar lipid-bilayer technique. Investigation of single-channel recordings revealed that LCC1 is voltage gated and strongly rectifying. In symmetrical 50 mm CaCl2 solutions, the single-channel conductance was 24 picosiemens. LCC1 showed a moderate selectivity for Ca2+ over K+ (9.4:1) and was permeable for a range of divalent cations (Ca2+, Ba2+, and Sr2+). In contrast to Bryonia dioica Ca2+ channel 1, a Ca2+-selective channel from the endoplasmic reticulum of touch-sensitive tendrils, LCC1 showed no bursting channel activity and had a low open probability and mean open time (2.83 ms at 50 mV). Inhibitor studies demonstrated that LCC1 is blocked by micromolar concentrations of erythrosin B (inhibitor concentration for 50% inhibition [IC50] = 1.8 μm) and the trivalent cations La3+ (IC50 = 5 μm) and Gd3+ (IC50 = 10 μm), whereas verapamil showed no blocking effect. LCC1 may play an important role in the regulation of the cytoplasmic free Ca2+ concentration in root-tip and/or root-cap cells. The question of whether this ion channel is part of the gravitropic signal transduction pathway deserves further investigation.

Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors

ATP-gated P2X2 receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X2 receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X2-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X2-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X2-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X2 receptor distribution on the seconds time scale.

A missense mutation of the Na+ channel αII subunit gene Nav1.2 in a patient with febrile and afebrile seizures causes channel dysfunction

Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in β1 and αI-subunit genes of voltage-gated Na+ channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R187W) in the gene encoding the α-subunit of neuronal voltage-gated Na+ channel type II (Nav1.2) in a patient with FS associated with afebrile seizures. The mutation R187W occurring on Arg187, a highly conserved residue among voltage-gated Na+ channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNav1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na+ channel conductance was not affected. Prolonged residence in the open state of the R187W mutant channel may augment Na+ influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Nav1.2 in a human disease and propose the R187W mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.