Functional Differences in Ionic Regulation between Alternatively Spliced Isoforms of the Na+-Ca2+ Exchanger from Drosophila melanogaster

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na⁺-Ca²⁺ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na⁺-Ca²⁺ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na⁺-Ca²⁺ exchange currents, where pipette Ca²⁺_o exchanges for bath Na⁺_i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na⁺_i affinities and current–voltage relationships, significant differences were observed in their Na⁺_i- and Ca²⁺_i-dependent regulatory properties. Both isoforms underwent Na⁺_i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na⁺_i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na⁺_i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na⁺-Ca²⁺ exchange activity by Ca²⁺_i, but their responses to regulatory Ca²⁺_i differed markedly. For both isoforms, the application of cytoplasmic Ca²⁺_i led to a decrease in outward exchange currents. This negative regulation by Ca²⁺_i is unique to Na⁺-Ca²⁺ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca²⁺ for all other characterized Na⁺-Ca²⁺ exchangers. For CALX1.1, Ca²⁺_i inhibited peak and steady state currents almost equally, with the extent of inhibition being ≈80%. In comparison, the effects of regulatory Ca²⁺_i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced (≈20–40% inhibition). For both exchangers, the effects of regulatory Ca²⁺_i occurred by a direct mechanism and indirectly through effects on Na⁺_i-induced inactivation. Our results show that regulatory Ca²⁺_i decreases Na⁺_i-induced inactivation of CALX1.2, whereas it stabilizes the Na⁺_i-induced inactive state of CALX1.1. These effects of Ca²⁺_i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na⁺-Ca²⁺ exchange proteins.     

Role of nova‐1 in regulating α2N,a novel glycine receptor splice variant,in developing spinal cord neurons

Inhibitory glycine receptor (GlyR) subunits undergo developmental regulation, but the molecular mechanisms of GlyR regulation in developing neurons are little understood. Using RT‐PCR, we investigated the regulation of GlyR α‐subunit splice forms during the development of the spinal cord of the rat. Experiments to compare the amounts of mRNA for two known splice variants of the GlyR α2 subunit, α2A and α2B, in the developing rat spinal cord revealed the presence of an additional, novel variant that lacked any exon 3, herein named “α2N.” Examination of the RNA from spinal cords of different‐aged rats showed a dramatic down‐regulation of α2N during prenatal development: α2N mRNA formed a significant portion of the α2 subunit pool at E14, but its relative level was reduced by 85% by birth and was undetectable in adults. Two proteins previously implicated in regulating the splicing of GlyR α2 pre‐mRNA, the neurooncological ventral antigen‐1 (Nova‐1) and the brain isoform of the polypyrimidine tract binding protein (brPTB), underwent small changes over the same period that did not correlate directly with the changes in the level of α2N, calling into question their involvement in the developmental regulation of α2N. However, treatment of spinal cord neurons in culture with antisense oligonucleotides designed selectively to knock down one of three Nova‐1 variants significantly altered the relative level of GlyR α2N, showing that Nova‐1 isoforms can regulate GlyR α2 pre‐mRNA splicing in developing neurons. These results provide evidence for a novel splice variant of the GlyR α2 subunit that undergoes dramatic developmental regulation, reveal the expression profiles of Nova‐1 and brPTB in the developing spinal cord, and suggest that Nova‐1 plays a role in regulating GlyR α2N in developing neurons. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 156–165, 2002     

Calcium channel γ subunits provide insights into the evolution of this gene family

The γ subunits of voltage-dependent calcium channels influence calcium current properties and may be involved in other physiological functions. Five distinct γ subunits have been described from human and/or mouse. The first identified member of this group of proteins, γ₁, is a component of the L-type calcium channel expressed in skeletal muscle. A second member, γ₂, identified from the stargazer mouse regulates the targeting of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors to the postsynaptic membrane. We report here the identification of three novel γ subunits from rat and mouse as well as the unidentified rat, mouse and human orthologs of the previously described subunits. Phylogenetic analysis of the 24 mammalian γ subunits suggests the following relationship ((((γ₂, γ₃), (γ₄, γ₈)), (γ₅, γ₇)), (γ₁, γ₆)) that indicates that they evolved from a common ancestral γ subunit via gene duplication. Our analysis reveals that the novel γ subunit γ₆ most closely resembles γ₁ and shares with it the lack of a PSD-95/DLG/ZO-1 (PDZ)-binding motif that is characteristic of most other γ subunits. Rat γ subunit mRNAs are expressed in multiple tissues including brain, heart, lung, and testis. The expression of γ₁ mRNA and the long isoform of γ₆ mRNA is most robust in skeletal muscle, while γ₆ is also highly expressed in cardiac muscle. Based on our analysis of the molecular evolution, primary structure, and tissue distribution of the γ subunits, we propose that γ₁ and γ₆ may share common physiological functions distinct from the other homologous γ subunits.     

The conserved dinucleotide AG of the 3′ splice site may be recognized twice during in vitro splicing of mammalian mRNA precursors

We have previously used site-directed mutagenesis to introduce an additional branch site into the first intron of the human β-globin gene at nt −24 between the natural branch site (nt−37) and the normal 3′ splice site (nt−1). We found that either the upstream or downstream branch site could be used during in vitro splicing, depending on which site best matched the mammalian branch site consensus YURAC (R = purine; Y = pyrimidine). Here we show that introduction of an additional AG dinucleotide at nt −20 between the downstream branch site and the normal 3′ splice site results in alternative 3′ splicing. Splicing to the new AG uses the upstream branch site exclusively, presumably because the downstream branch site is only 4 nt from this 3′ splice site. We were surprised, however, to find that the presence of the new AG also prevents the use of the upstream branch site for splicing to the normal 3′ splice site. Analysis of additional mutants confirmed earlier work [Krainer et al.: Mechanisms of human β-globin pre-mRNA splicing. In Berg, P. (Ed.), The Robert A. Welch Foundation Conferences on Chemical Research XXIX. Genetic Chemistry: The Molecular Basis of Heredity. Welch Foundation, Houston, TX, 1985, pp. 353–382] that the new AG cannot function by itself as a complete 3′ splice site; rather, it appears that alternative 3′ splicing initiates at the normal 3′ splices site but then searches, once the reaction is underway, for the first AG downstream from the chosen branch site. Taken together, our data suggest that the conserved AG dinucleotide at the 3′ splice site may be recognized twice during mammalian mRNA splicing in vitro.     

Characterization of the 5′-to-5′linked adult α- and β-globin genes from three sciaenid fish species (Pseudosciaena crocea, Sciaenops ocellatus, Nibea miichthioides)

Recently, we cloned the adult α-globin genes from large yellow croaker Pseudosciaena crocea, cuneate drum Nibea miichthioides and red drum Sciaenops ocellatus. All these α-globins have a unique Gly insertion at the 47th residue. In this paper, the three sciaenid globin complexes were identified and compared in detail. Linkage analysis indicated that the sciaenid α- and β-globin genes were oriented head-to-head relative to each other. The sciaenid intergenic regions between the linked α- and β-globin genes were the smallest in reported fish globin gene complexes to date. Classical promoter elements were condensed and the CCAAT box unstable duplication was found in these regions. The promoter function of the intergenic region from large yellow croaker was tested by transient expression of EGFP in Vero cells. We also described a method for studying luciferase reporter gene transient expression in primary fish erythrocytes. We used the method to assess the promoter strength of the three intergenic regions between the sciaenid α- and β-globin genes.     

Enantioselective syntheses and calcium channel modulating effects of (+)- and (−)-3-isopropyl 5-(4-methylphenethyl) 1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)-3,5-pyridinedicarboxylates

The (+)- and (?)-enantiomers of 3-isopropyl 5-(4-methylphenethyl) 1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)-3,5-pyridinedicarboxylate were synthesized using an efficient highly enantioselective (ee ≥ 96%) variant of the Hantzsch dihydropyridine synthesis. The key step in this procedure involved the asymmetric Michael addition of a metalated chiral aminocrotonate, derived from D -valine or L -valine, respectively, to the Knoevenagel acceptor (Z)-2-isopropoxycarbonyl-1-(2-pyridyl)-but-1-en-3-one. Both enantiomers exhibited a dual cardioselective partial calcium channel agonist (positive inotropic)/smooth muscle selective calcium channel antagonist effect. The relative in vitro smooth muscle calcium channel antagonist activities of the (?):(+) enantiomers was 26:1. In contrast, the (+)-enantiomer exhibited a greater in vitro positive inotropic effect on guinea pig left atrium where the contractile force was maximally increased by 14.8% at a concentration of 1.63 × 10^?8 M. © 1994 Wiley-Liss, Inc.     

Cis-acting elements regulate alternative splicing of exons 6A, 6B and 8 of the α1(XI) collagen gene and contribute to the regional diversification of collagen XI matrices

Consecutive exons 6A, 6B, 7 and 8 that encode the variable region of the amino-terminal domain (NTD) of the col11a1 gene product undergo a complex pattern of alternative splicing that is both tissue-dependent and developmentally regulated. Expression of col11a1 is predominantly associated with cartilage where it plays a critical role in skeletal development. At least five splice-forms (6B-7-8, 6A-7-8, 7-8, 6B-7 and 7) are found in cartilage. Splice-forms containing exon 6B or 8 have distinct distributions in the long bone during development, while in non-cartilage tissues, splice-form 6A-7-8 is typically expressed. In order to study this complex and tissue-specific alternative splicing, a mini-gene that contains mouse genomic sequence from exon 5 to 11, flanking the variable region of α1(XI)-NTD, was constructed. The minigene was transfected into chondrocytic (RCS) and non-chondrocytic (A204) cell lines that endogenously express α1(XI), as well as 293 cells which do not express α1(XI). Alternative splicing in RCS and A204 cells reflected the appropriate cartilage and non-cartilage patterns while 293 cells produced only 6A-7-8. This suggests that 6A-7-8 is the default splicing pathway and that cell or tissue-specific trans-acting factors are required to obtain pattern of the alternative splicing of α1(XI) pre-mRNA observed in chondrocytes. Deletional analysis was used to identify cis-acting regions important for regulating splicing. The presence of the intact exon 7 was required to generate the full complex chondrocytic pattern of splicing. Furthermore, deletional mapping of exon 6B identified sequences required for expression of exon 6B in RCS cells and these may correspond to purine-rich (ESE) and AC-rich (ACE) exonic splicing enhancers.