The promiscuous role of the epsilon subunit in GABA(A) receptor biogenesis

The formation of alpha1beta2gamma2epsilon receptors suggests that the epsilon subunit does not displace the single gamma2 subunit in alpha1beta2gamma2 receptors. Thus, epsilon must replace alpha and/or beta subunit(s) if the pentameric receptor structure is to be preserved. To assess the potential for which subunit is replaced in alphabetaepsilon and alphabetagammaepsilon receptors we analyzed the assembly and functional expression of the epsilon subunit with respect to alpha1, beta2 and gamma2 subunits. Using concatenated subunits, we have determined that epsilon is capable of substituting for either (but not both) of the alpha subunits, one of the beta subunits, and possibly the gamma2 subunit. However, the most likely sites at which the epsilon subunit may contribute to receptor function appears to be at position 1 (replaces alpha1) in alphabetagammaepsilon (varepsilon-beta2-alpha1-beta2-gamma2) receptors, or at position 4 (replaces beta2) in alphabetaepsilon (alpha1-beta2-alpha1-varepsilon-beta2) receptors. In both cases, it appears that only a single GABA binding site is present.

Increased expression of the auxiliary beta2-subunit of ventricular L-type Ca2+ channels leads to single-channel activity characteristic of heart failure

BACKGROUND: Increased activity of single ventricular L-type Ca(2+)-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation. METHODS AND RESULTS: By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V)1.2 also reveal increased single-channel activity and sarcolemmal beta(2) expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression. Additional evidence for the cause-effect relationship between beta(2)-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V)1.2 and inducible beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(2)-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. CONCLUSIONS: Our study presents evidence of the pathobiochemical relevance of beta(2)-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Sudden infant death syndrome-associated mutations in the sodium channel beta subunits.

BACKGROUND Approximately 10% of sudden infant death syndrome (SIDS) cases may stem from potentially lethal cardiac channelopathies, with approximately half of channelopathic SIDS involving the Na(V)1.5 cardiac sodium channel. Recently, Na(V) beta subunits have been implicated in various cardiac arrhythmias. Thus, the 4 genes encoding Na(V) beta subunits represent plausible candidate genes for SIDS. OBJECTIVE This study sought to determine the spectrum, prevalence, and functional consequences of sodium channel beta-subunit mutations in a SIDS cohort. METHODS In this institutional review board-approved study, mutational analysis of the 4 beta-subunit genes, SCN1B to 4B, was performed using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing of DNA derived from 292 SIDS cases. Engineered mutations were coexpressed with SCN5A in HEK 293 cells and were whole-cell patch clamped. One of the putative SIDS-associated mutations was similarly studied in adenovirally transduced adult rat ventricular myocytes. RESULTS Three rare (absent in 200 to 800 reference alleles) missense mutations (beta3-V36M, beta3-V54G, and beta4-S206L) were identified in 3 of 292 SIDS cases. Compared with SCN5A+beta3-WT, beta3-V36M significantly decreased peak I(Na) and increased late I(Na), whereas beta3-V54G resulted in a marked loss of function. beta4-S206L accentuated late I(Na) and positively shifted the midpoint of inactivation compared with SCN5A+beta4-WT. In native cardiomyocytes, beta4-S206L accentuated late I(Na) and increased the ventricular action potential duration compared with beta4-WT. CONCLUSION This study provides the first molecular and functional evidence to implicate the Na(V) beta subunits in SIDS pathogenesis. Altered Na(V)1.5 sodium channel function due to beta-subunit mutations may account for the molecular pathogenic mechanism underlying approximately 1% of SIDS cases.

Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase.

Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).

Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.

gamma-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are constructed from four subunit classes with multiple members: alpha (1-6), beta (1-4), gamma (1-4), and delta (1). The contribution of subunit diversity in determining receptor subcellular targeting was examined in polarized Madin-Darby canine kidney (MDCK) cells. Significant detection of cell surface homomeric receptor expression by a combination of both immunological and electrophysiological methodologies was only found for the beta 3 subunit. Expression of alpha/beta binary combinations resulted in a nonpolarized distribution for alpha 1 beta 1 complexes, but specific basolateral targeting of both alpha 1 beta 2 and alpha 1 beta 3 complexes. The polarized distribution of these alpha/beta complexes was unaffected by the presence of the gamma 2S subunit. Interestingly, delivery of receptors containing the beta 3 subunit to the basolateral domain occurs via the apical surface. These results show that beta subunits can selectively target GABAA receptors to distinct cellular locations. Changes in the spatial and temporal expression of beta-subunit isoforms may therefore provide a mechanism for relocating GABAA receptor function between distinct neuronal domains. Given the critical role of these receptors in mediating synaptic inhibition, the contribution of different beta subunits in GABAA receptor function, may have implications in neuronal development and for receptor localization/clustering.

Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta.

The alpha subunit of the karyopherin heterodimer functions in recognition of the protein import substrate and the beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of karyopherin beta's binding sites both for Ran-GTP and for karyopherin alpha. We discovered that karyopherin beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and karyopherin alpha bind to overlapping sites on karyopherin beta. Having a higher affinity to the overlapping site, Ran-GTP displaces karyopherin alpha and binds to karyopherin beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for karyopherin beta to a cluster of basic residues analogous to those previously shown to constitute karyopherin alpha's binding site to karyopherin beta.

A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.

Mutations in the gene encoding the alpha subunit of the rod cGMP-gated channel in autosomal recessive retinitis pigmentosa.

Mutations in the genes encoding two proteins of the retinal rod phototransduction cascade, opsin and the beta subunit of rod cGMP phosphodiesterase, cause retinitis pigmentosa (RP) in some families. Here we report defects in a third member of this biochemical pathway in still other patients with this disease. We screened 94 unrelated patients with autosomal dominant RP and 173 unrelated patients with autosomal recessive RP for mutations in the gene encoding the alpha subunit of the rod cGMP-gated cation channel. Five mutant sequences cosegregated with disease among four unrelated families with autosomal recessive RP. Two of these were nonsense mutations early in the reading frame (Glu76End and Lys139End) and one was a deletion encompassing most if not all of the transcriptional unit; these three alleles would not be expected to encode a functional channel. The remaining two mutations were a missense mutation (Ser316Phe) and a frameshift [Arg654(1-bp del)] mutation truncating the last 32 aa in the C terminus. The latter two mutations were expressed in vitro and found to encode proteins that were predominantly retained inside the cell instead of being targeted to the plasma membrane. We conclude that the absence or paucity of functional cGMP-gated cation channels in the plasma membrane is deleterious to rod photoreceptors and is an uncommon cause of RP.

Membrane disposition of the M5-M6 hairpin of Na+,K(+)-ATPase alpha subunit is ligand dependent.

Extensive proteolytic digestion of Na+,K(+)-ATPase (EC 3.6.1.37) by trypsin produces a preparation where most of the extramembrane portions of the alpha subunit have been digested away and the beta subunit remains essentially intact. The fragment Gln-737-Arg-829 of the Na+,K(+)-ATPase alpha subunit, which includes the putative transmembrane hairpin M5-M6, is readily, selectively, and irreversibly released from the posttryptic membrane preparation after incubation at 37 degrees C for several minutes. Once released from the membrane, the fragment aggregates but remains water soluble. Occlusion of K+ or Rb+ specifically prevents release of the Gln-737-Arg-829 fragment into the supernatant. Labeling of the posttryptic membrane preparation with cysteine-directed reagents revealed that Cys-802 (which is thought to be located within the M6 segment) is protected against the modification by Rb+ while this fragment is in the membrane but can be readily modified upon release. Cation occlusion apparently alters the folding and/or disposition of the M5-M6 fragment in the membrane in a way that does not occur when the fragment migrates to the aqueous phase. The ligand-dependent disposition of the M5-M6 hairpin in the membrane along with recent labeling studies suggest a key role for this segment in cation pumping by Na+,K(+)-ATPase.

NusA interferes with interactions between the nascent RNA and the C-terminal domain of the alpha subunit of RNA polymerase in Escherichia coli transcription complexes.

The effects of NusA on the RNA polymerase contacts made by nucleotides at internal positions in the nascent RNA in Escherichia coli transcription complexes were analyzed by using the photocrosslinking nucleotide analog 5-[(4-azidophenacyl) thio]-UMP. It was placed at nucleotides between +6 and +15 in RNA transcribed from the phage lambda PR' promoter. Crosslinks of analog in these positions in RNAs which contained either 15, 28, 29, or 49 nt were examined. Contacts between the nascent RNA and proteins in the transcription complex were analyzed as the RNA was elongated, by placing the crosslinker nearest the 5' end of the RNA 10, 23, 24, or 44 nt away from the 3' end. The beta or beta' subunit of polymerase, and NusA when added, were contacted by RNA from 15 to 49 nt long. When the upstream crosslinker was 24 nt from the 3" end of the RNA (29-nt RNA), alpha was also contacted in the absence of NusA. The addition of NusA prevented RNA crosslinking to alpha. When the crosslinker was 44 nt from the 3' end (49-nt RNA), alpha crosslinks were still observed, but crosslinks to beta or beta' and NusA were greatly diminished. RNA crosslinking to alpha, and loss of this crosslink when NusA was added, was observed in the presence of NusB, NusE, and NusG and when transcription was carried out in the presence of an E. coli S100 cell extract. Peptide mapping localized the RNA interactions to the C-terminal domain of alpha.

Inhibition of protein interactions with the beta(2) sliding clamp of Escherichia coli DNA polymerase III by peptides from beta(2)-binding proteins

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cells

The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel beta and alpha(2)-delta subunits on expression levels and biophysical properties of three different types (Ca(v)1.2, Ca(v)2.1 and Ca(v)2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Ca(v)1.2 and Ca(v)2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel beta subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel alpha(1) subunit, and beta(3) subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the alpha(2)-delta subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Ca(v)2 channel family, appears to act synergistically with beta subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by beta and by alpha(2)-delta subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the alpha(2)-delta(1) subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes.

Overexpressed Ca-v beta 3 inhibits N-type (Ca(v)2.2) calcium channel currents through a hyperpolarizing shift of "ultra-slow" and "closed-state" inactivation

It has been shown that P auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a hovel inhibitory effect of beta3 Subunit on macroscopic Ba2+ currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/ cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at physiological holding potentials (HPs) of -60 and -80 mV At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca,.2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced Current suppression was reversed at a HP of - 120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba-2divided by (40 mM) as a charge carrier also largely diminished the effect of P3 at -80 mV Therefore, experimental conditions (HP, divalent cation concentration, and P3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel P3 effect. Steady-state inactivation curves revealed that N-type channels exhibited closed-state inactivation without P3, and that P3 caused an similar to40 mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of P3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV Similar ultra-slow inactivation was observed for N-type channels Without P3. Thus, P3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by Causing a hyperpolarizing shift of the inactivation without affecting ultra-slow and closed-state inactivation properties.

Solution structure of the human signaling protein RACK1

Background: The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development. Results: In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 +/- 0.2) x 10(6) M(-1) and resulted in a dissociation constant (KD) of (0.7 +/- 0.1) x 10(-6) M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion. Conclusion: Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.