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M C Emerick

Publications and source records attributed to M C Emerick.

8 recordsLinked to original sources
PubMed2000

Full-length single-gene cDNA libraries: applications in splice variant analysis.

Alternative splicing of pre-mRNA may generate many distinct proteins from a single gene: regulation of alternative exon selection constitutes control of molecular structure downstream of transcription. Identifying natural splice variants among hundreds or thousands of theoretical alternatives, and examining the regulation of exon selection at multiple sites, may require screening many full-length cDNAs. We describe methods for preparing full-length cDNA libraries comprising the splice variants from single genes. The methods employ robust long distance reverse transcription, gene-specific second strand synthesis, long PCR, and cloning: with these methods cDNAs coding full-length open reading frames were prepared for 21 ion channels (1.2-15 kb). Exon combinations in isolated clones are determined by multiplex PCR. Approximately 85% of the clones contain full-length inserts. Screening can detect even rare variants (0.1%) in linear proportion to their abundance in initial mRNA pools. Tissue-specific expression patterns are reproducible. We describe methods for quantifying and minimizing artifactual exon recombination by template switching. These methods can be used to generate thousands of full-length clones of even large transcripts (>8 kb) for the systematic identification of splice variants and the analysis of regulation of alternative exon selection.

Alternative Splicing↗
PubMed1999

Structure and alternative splicing of the gene encoding alpha1I, a human brain T calcium channel alpha1 subunit.

The structure of CACNA1I, the gene encoding alpha1I, a human brain T Ca2+ channel alpha1 subunit, was determined by comparison of polymerase chain reaction-amplified brain cDNA and genomic sequences. The gene consists of at least 36 exons spanning at least 115,168 basepairs of chromosome 22q12.3-13.2. The predicted protein has 2016 amino acids and 28 potential phosphorylation sites. Alternative splicing of the gene occurs at two sites: cassette exon 9 and an alternative acceptor in exon 33. Molecular diversity generated by alternative splicing and post-translational modification of this and other members of the T alpha1 subunit gene family may account for the observed heterogeneity of T currents in central neurons.

Alternative Splicing↗
PubMed1993

Intramolecular fusion of Na pump subunits assures exclusive assembly of the fused alpha and beta subunit domains into a functional enzyme in cells also expressing endogenous Na pump subunits.

Experiments designed to identify Na pump structural features which tag the molecule for asymmetric cell-surface localization are inherently complex because either subunit, or both, may contain targeting information and because the cells which recognize those targeting signals and maintain asymmetric plasma membrane domains also express their own Na pumps, the subunits of which can assemble into hybrid pump molecules with pump subunits expressed from transfected cDNA clones. Cotransfecting cDNA for both subunits only complicates matters further by resulting in expression of four distinct dimeric molecular species. To eliminate the potential for cross-assembly in these and other experiments we have constructed cDNA encoding a "single-subunit" Na pump (called "alpha-beta") in which the alpha and beta subunits are joined by a linker of 17 amino acids. By all criteria tested alpha-beta functioned as a normal heterodimeric Na pump. It was expressed in a variety of mammalian cell lines as a single, high molecular weight polypeptide located primarily on the surface membrane, with the beta subunit exposed to the extracellular medium. Binding of the conformation-sensitive monoclonal antibody 24 to the beta subunit indicated that the fusion protein was folded as a properly "assembled" sodium pump. Expression of alpha-beta in ouabain-resistant mouse L cells resulted in high affinity ouabain binding and ouabain-sensitive, sodium-dependent rubidium transport. The enzyme was properly targeted to the basolateral plasma membrane in polarized epithelial cells. The functional integrity of the fusion protein renders it suitable for site-directed mutagenesis studies of targeting and enzymology where control of subunit assembly is desired. These results also support topological models in which the carboxyl terminus of the alpha subunit is cytoplasmic.

Amino Acid Sequence↗
PubMed1993

Regulation of the eel electroplax Na channel and phosphorylation of residues on amino- and carboxyl-terminal domains by cAMP-dependent protein kinase.

Previous studies have shown that the short-motif electroplax Na channel is phosphorylated in vitro by cyclic AMP-dependent protein kinase (PKA) at serines 6 or 7 and 1776 and threonine 17 (Emerick & Agnew, 1989). We here show that phosphatase treatment of solubilized, purified Na channels enhanced subsequent PKA labeling of four of five tryptic phosphopeptides, indicating that these sites are phosphorylated in vivo. Microsequencing and analysis of PTH-amino acid products revealed endogenous labeling of serines 6, 444, 1680, and 1776. Serines 1680 and 1776 lie in the carboxyl-terminal cytoplasmic domain, while serine 6 lies in the amino terminus and serine 444 is in the cytoplasmic loop between domains I and II. Endogenous phosphorylation of serine 6 establishes experimentally that the Na channel amino terminus is cytoplasmic. In electrophysiological experiments, brief exposure of inside-out membrane patches excised from Sachs-organ cells to MgATP and purified PKA catalytic subunit produced rapid, sustained reduction of Na current amplitude by approximately 80% and a hyperpolarizing shift in the conductance/voltage relation by 10-12 mV. The effect was absent in controls omitting either PKA or MgATP. Serines 6 and 1776 and threonine 17 are labeled rapidly and extensively in vitro, and only threonine 17 appears to be unphosphorylated in vivo. We suggest that phosphorylation of the amino and carboxyl domains, perhaps especially at threonine 17, underlies the demonstrated downregulation of the electroplax Na channel.

Animals↗
PubMed1989

Identification of phosphorylation sites for adenosine 3',5'-cyclic phosphate dependent protein kinase on the voltage-sensitive sodium channel from Electrophorus electricus.

The voltage-sensitive sodium channel from the electroplax of Electrophorus electricus is selectively phosphorylated by the catalytic subunit of cyclic-AMP-dependent protein kinase (protein kinase A) but not by protein kinase C. Under identical limiting conditions, the protein was phosphorylated 20% as rapidly as the synthetic model substrate kemptamide. A maximum of 1.7 +/- 0.6 equiv of phosphate is incorporated per mole. Phosphoamino acid analysis revealed labeled phosphoserine and phosphothreonine at a constant ratio of 3.3:1. Seven distinct phosphopeptides were identified among tryptic fragments prepared from radiolabeled, affinity-purified protein and resolved by HPLC. The three most rapidly labeled fragments were further purified and sequenced. Four phosphorylated amino acids were identified deriving from three consensus phosphorylation sites. These were serine 6, serine 7, and threonine 17 from the amino terminus and a residue within 47 amino acids of the carboxyl terminus, apparently serine 1776. The alpha-subunits of brain sodium channels, like the electroplax protein, are readily phosphorylated by protein kinase A. However, these are also phosphorylated by protein kinase C and exhibit a markedly different pattern of incorporation. Each of three brain alpha-subunits displays an approximately 200 amino acid segment between homologous repeat domains I and II, which is missing from the electroplax and skeletal muscle proteins [Noda et al. (1986) Nature (London) 320, 188; Kayano et al. (1988) FEBS Lett. 228, 1878; Trimmer et al. (1989) Neuron 3, 33]. Most of the phosphorylation of the brain proteins occurs on a cluster of consensus phosphorylation sites located in this segment. This contrasts with the pattern of highly active sites on the amino and carboxyl termini of the electroplax protein. The detection of seven labeled tryptic phosphopeptides compared to the maximal labeling stoichiometry of approximately 2 suggests that many of the acceptor sites on the protein may be blocked by endogenous phosphorylation.

Amino Acid Sequence↗
PubMed1989

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid.

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including alpha-(2----8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis alpha-(2----8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3200 pmol of [3H]TTX-binding sites/mg of protein and a single polypeptide of approximately 285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. We further describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

Animals↗
PubMed1986

Fluorescence assay for neurotoxin-modulated ion transport by the reconstituted voltage-activated sodium channel isolated from eel electric organ.

A fluorescence assay for measuring Na channel activation in liposomes containing voltage-sensitive Na channels isolated from Electrophorus electricus is described. The assay is based on transport of a heavy-metal cation, T1+, through the activated channel to quench fluorescence of an internalized, water-soluble chromophore. The channel is "locked" in a chronically opened configuration with alkaloid neurotoxins such as veratridine or batrachotoxin. Diffusion potentials are used to amplify the signal, and enlarged liposomes (greater than 8000 A) result in time courses extended to the range of seconds. Analysis of the kinetics of quenching yields parameters that behave as linear functions of channel activation and reflect vesicle size and channel abundance. The k1/2's for activation by veratridine and batrachotoxin were 5 microM and 169 nM, respectively, and that for tetrodotoxin blockade was 4 nM. Externally applied QX-222 and tetrodotoxin each acted to partially block the stimulated signal, as expected for compounds that act on oppositely oriented channels in the membrane. Single-channel conductances estimated with either veratridine or batrachotoxin ranged between 0.6 and 40.7 pS, corresponding to transport numbers of (1.2 X 10(5)) to (8.1 X 10(6)) ions s-1 channel-1 under the conditions of assay. The assay is approximately 100-fold more sensitive than radiotracer influx assays, requiring 1 fmol of protein per time course.

Animals↗