Shaker K+ channel subunits from heteromultimeric channels with novel functional properties.

A large number of related genes (the Sh gene family) encode potassium channel subunits which form voltage-dependent K+ channels by aggregating into homomulitimers. One of these genes, the Shaker gene in Drosophila, generates several products by alternative splicing. These products encode proteins with a constant central region flanked by variable amino and carboxyl domains. Coinjection of two Shaker RNAs with different amino or different carboxyl ends into Xenopus oocytes produces K+ currents that display functional properties distinct from those observed when each RNA is injected separately, indicating the formation of heteromultimeric channels. The analysis of Shaker heteromultimers suggests certain rules regarding the roles of variable amino and carboxyl domains in determining kinetic properties of heteromultimeric channels. Heteromultimers with different amino ends produce currents in which the amino end that produces more inactivation dominates the kinetics. In contrast, heteromultimers with different carboxyl ends recover from inactivation at a rate closer to that observed in homomultimers of the subunit which results in faster recovery. While this and other recent reports demonstrate that closely related Sh family proteins form functional heteromultimers, we show here that two less closely related Sh proteins do not seem to form functional heteromultimeric channels. The data suggest that sites for subunit recognition may be found in sequences within a core region, starting about 130 residues before the first membrane spanning domain of Shaker and ending after the last membrane spanning domain, which are not conserved between Sh Class I and Class III genes.     

Three functional isoforms of GAR-2, a Caenorhabditis elegans G-protein-linked acetylcholine receptor, are produced by alternative splicing.

We have previously isolated a cDNA clone from Caenorhabditis elegans that encodes a novel form of G-protein-linked acetylcholine receptor, termed GAR-2. GAR-2 is similar to but pharmacologically distinct from muscarinic acetylcholine receptors. Here we report the identification of two gar-2 cDNA clones that are different from the previous one. These newly identified cDNAs encode polypeptides of 664 and 627 amino acids, whereas the previous one encodes a polypeptide of 614 amino acids. The three GAR-2 isoforms, which differ only in the third intracellular loop, arise from alternative splicing. Electrophysiological analyses using the Xenopus oocyte system showed that all three GAR-2 isoforms couple to the activation of G-protein-gated inwardly rectifying K+ (GIRK1) channel with similar drug specificity. Our results indicate that alternative splicing plays an important role in promoting molecular diversity of G-protein-linked acetylcholine receptors in C. elegans.     

Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain.

Cloning and sequencing of cDNAs isolated from a rat cortex cDNA library reveals that a gene family encodes several highly homologous K+ channel forming (RCK) proteins. Functional characterization of the channels expressed in Xenopus laevis oocytes following microinjection of in vitro transcribed RCK-specific RNAs shows that each of the RCK proteins forms K+ channels that differ greatly in both their functional and pharmacological properties. This suggests that the molecular basis for the diversity of voltage-gated K+ channels in mammalian brain is based, at least partly, on the expression of several RCK proteins by a family of genes and their assembly to homooligomeric K+ channels with different functional properties.     

Level of expression controls modes of gating of a K+ channel.

Several distinct subfamilies of K+ channel genes have been discovered by molecular cloning, however, in some cases the structural differences among them do not account for the diversity of K+ current types, ranging from transient A-type to slowly inactivating delayed rectifier-type, as members within each subfamily have been shown to code for K+ channels of different inactivation kinetics and pharmacological properties. We show that a single K+ channel cDNA of the Shaker subfamily (ShH4) can express in Xenopus oocytes not only a transient A-type K+ current but also, upon increased level of expression, slowly inactivating K+ currents with markedly reduced sensitivity to tetraethylammonium. In correlation with the macroscopic currents there are single-channel gating modes ranging from the fast-inactivation mode which underlies the transient A-type current, to slow-inactivation modes characterized by bursts of longer openings, and corresponding to the slowly inactivating macroscopic currents.     

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.     

Isolation of multiple mouse cDNAs with coding homology to Saccharomyces cerevisiae CDC25: identification of a region related to Bcr, Vav, Dbl and CDC24.

In Saccharomyces cerevisiae, the product of the CDC25 gene is an essential Ras activator that appears to function by stimulating guanine nucleotide exchange on Ras. Using the ability of a mouse cDNA expression library to complement yeast cells lacking functional CDC25, Martegani et al. have identified a 1.7 kb partial cDNA from a gene, designated CDC25Mm, with homology to CDC25. We have now screened a mouse brain cDNA library to identify full-length clones of CDC25Mm. This cloning has led to the isolation of six distinct full-length cDNAs, each of which appear to be derived from the CDC25Mm gene, since their 3' 2 kb appear to be identical and to encode the same 661 C-terminal amino acids. Three cDNAs are predicted to encode protein products of 666 or 667 amino acids. The other three cDNAs encode products that are 836, 1120 and 1260 amino acids, respectively. A 241 amino acid region near the N-terminus of the two largest products was found to have homology to a domain shared by Bcr, Vav, Dbl and CDC24. Polyclonal antibodies raised to a peptide encoded by all the cDNAs have identified at least two protein products in NIH3T3 fibroblasts. Their apparent molecular weights are 75 and 95 kDa, which correspond closely to those predicted to be encoded, respectively, by the two shorter classes of cDNAs. In NIH3T3, the 95 kDa form is much more abundant than the 75 kDa form, while PC-12 pheochromocytoma cells contain relatively high levels of the 75 kDa form. We conclude that CDC25Mm is a complex gene whose protein products are regulated in a tissue-specific manner.     

Epstein-Barr virus mRNAs produced by alternative splicing.

The structure of Epstein-Barr virus mRNAs transcribed in B95-8 cells has been studied by cDNA cloning and sequencing. We present here the analysis of four cDNAs. The corresponding mRNAs are probably transcribed from a single promoter located in the US region. They are produced by alternative splicing of exons transcribed from the US, IR and UL regions. The exons are spread over 100 kbp. The exons from the IR region constitute a unit which is repeated several times. The cDNAs share the exons from the US and IR regions. Some of the cDNAs also share some of the exons from the UL region. Each cDNA contains a long open reading frame or the 5' end of a long open reading frame which ends several hundred nucleotides downstream on the viral genome. The 5' untranslated regions are unusually long. Three mRNA species differing in their 5' untranslated regions may encode for the nuclear antigen EBNA-1. The other mRNAs encode for polypeptides which may not have any common region.     

The human sex hormone-binding globulin gene contains exons for androgen-binding protein and two other testicular messenger RNAs

When a sex hormone-binding globulin (SHBG) cDNA was used to screen a human testicular cDNA library, three distinct cDNAs were isolated, one of which corresponds to the human SHBG cDNA sequence and probably represents testicular androgen-binding protein. The other two SHBG-related cDNAs each contain unique 5' regions that diverge from the SHBG cDNA sequence at the same position, and one of them (SHBGr-2) lacks a 208-base pair region within the SHBG cDNA. As a result, this cDNA could potentially encode for a truncated form of SHBG which lacks N-linked carbohydrates and part of the steroid-binding domain. Southern blots of human placental DNA and cloned genomic DNA fragments also indicate that SHBG and its related testicular cDNAs are the products of a single gene. Sequence analysis of the gene indicates that the complete coding region for the SHBG precursor is comprised of 8 exons, which are distributed over 3.2 kilobase (kb) of genomic DNA, and the unique 5' regions associated with the two SHBG-related testicular cDNAs were identified 1.9 kb upstream from the initiating codon for SHBG. In addition, the deletion within SHBGr-2 is due to the removal of exon 7, and an interesting feature of the gene is that differentially used exons are preceded by Alu repetitive DNA sequences. Although the relative abundance of the various SHBG-related mRNAs in the testis has not been established, Northern blot analysis indicates that they are similar in size (1.6 kb) to that of hepatic SHBG mRNA.     

Multiple mRNA species code for the catalytic subunit of the cAMP-dependent protein kinase from LLC-PK1 cells. Evidence for two forms of the catalytic subunit

We present evidence for the existence of two forms of the catalytic (C) subunit of the cAMP-dependent protein kinase. A lambda gt-11 cDNA library constructed from poly(A)-rich RNA from the porcine kidney cell line, LLC-PK1, was screened using a 1.5-kb EcoRI fragment from a bovine cDNA for the C subunit. Two independent classes of cDNAs were identified on the basis of partial restriction map and sequence data. These two cDNAs, lambda CAT4 and lambda CAT3, apparently encode two forms of C subunit designated C alpha and C beta, respectively. The nucleotide sequence of the C alpha and C beta cDNAs revealed differences in the coding region and particularly in the 3' untranslated region. However, the deducted amino acid sequences of C alpha and C beta subunits were 96% homologous to the sequences so far determined. Specific probes from the 3' coding region of the two cDNA species were used to investigate C subunit mRNA expression in LLC-PK1 cells. Northern analysis showed a major mRNA species of 2.8 kb with the C alpha probe while the C beta probe detected two mRNA species of 5.0 kb and 3.8 kb. These data were supported by genomic blot analysis which showed distinct hybridization patterns with either the C alpha or C beta probes. All the available evidence suggests that at least two distinct genes encode the C subunit which are expressed in LLC-PK1 cells.     

A novel alpha subunit in rat brain GABAA receptors

Two cDNAs (alpha 1 and alpha 4) from rat brain cDNA libraries encode isoforms of the alpha subunit of the GABA/benzodiazepine receptor, which differ at 30% of their amino acid residues. Northern blot analysis and in situ hybridization histochemistry show that alpha 1 and alpha 4 mRNAs have distinct sizes and distinct regional and cellular distributions in rat brain: both mRNAs are found in the cortex and hippocampus; however, only the alpha 1 mRNA is detected in the cerebellum. We injected RNA transcribed from alpha 1 and alpha 4 cDNAs into Xenopus oocytes, together with an RNA for a rat beta subunit. We obtained GABA-dependent inward currents that were reversibly blocked by picrotoxin. Picrotoxin alone, applied to oocytes producing the alpha and beta polypeptides, elicited an outward current. We suggest that these polypeptides together produce GABA-gated ion channels that can also open spontaneously.     

A single gene codes for two forms of rat nucleolar protein B23 mRNA

Protein B23 (38 kDa, pI = 5.1) is an abundant RNA-associated nucleolar phosphoprotein and putative ribosome assembly factor. A full length cDNA clone (lambda JH1) encoding a major expressed form of rat protein B23, now designated B23.1, was reported recently (Chang, J. H., Dumbar, T. S., and Olson, M. O. J. (1988) J. Biol. Chem. 263, 12824-12837). In this paper the isolation from a rat brain library and sequence of a cDNA clone (lambda JH2) coding for a second form (B23.2) of protein B23 is reported. Isoforms B23.1 and B23.2 are polypeptides of 292 and 257 amino acids, respectively. The 5'-untranslated regions of the two cDNAs and the amino-terminal 255 amino acids of the proteins are identical in the two isoforms. However, the 3'-untranslated regions of the mRNAs are completely different, and the dipeptide Gly-Gly in B23.1 (residues 256 and 257) is replaced by Ala-His in B23.2 indicating that the former is not a precursor of the latter. The finding of AGGT sequences in the 3' regions of lambda JH1 suggest the presence of intron-exon boundaries at the point where the two cDNAs begin to differ. To investigate the origin of the two isoforms, two rat genomic libraries were screened with oligonucleotide probes based on sequences from the unique regions of the two cDNAs. One of the genomic clones isolated (lambda JH125) contained a 6.5-kilobase fragment encoding the 3' end of both cDNAs. lambda JH125 contains four exons designated W, X, Y, and Z in the order indicated. Exons W and X encode 36 amino acids at the carboxyl terminus of B23.2, whereas exons W, Y, and Z encode the carboxyl-terminal 71 amino acid residues of B23.1. Exons X and Z each contain distinct 3'-untranslated sequences in which are found polyadenylation signals. These data suggest that two different mRNAs are formed by alternative splicing of separate 3' segments onto a common 5' region.     

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels.

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

Secreted ferritin subunits are of two kinds in insects molecular cloning of cDNAs encoding two major subunits of secreted ferritin from Calpodes ethlius

In insects, holoferritin is easily visible in the vacuolar system of tissues that filter the hemolymph and, at least in Lepidoptera, is abundant in the hemolymph. Sequences reported for insect secreted ferritins from Lepidoptera and Diptera have high sequence diversity. We examined the nature of this diversity for the first time by analyzing sequences of cDNAs encoding two ferritin subunits from one species, Calpodes ethlius (Lepidoptera, Hesperiidae). We found that insect secreted ferritin subunits are of two types with little resemblance to each other. Ferritin was isolated from iron loaded hemolymph of C. ethlius fifth instar larvae by differential centrifugation. The N-terminal amino acid sequences for the nonglycosylated subunit with Mr 24,000 (S) and the largest glycosylated subunit with Mr 31,000 (G) were determined. The N-termini of the two subunits were different and were used to construct degenerate PCR primers. The same cDNA products were amplified from cDNA libraries from the midgut which secretes holoferritin and from the fat body which secretes iron-poor apoferritin. The G subunit most closely resembles the glycosylated ferritin subunit from Manduca sexta and the S subunit resembles the Drosophila small subunit. The S and G subunits from Calpodes were dissimilar and distinct from the cytosolic ferritins of vertebrates and invertebrates. Additional sequences were obtained by 5' and 3' RACE from separate fat body and midgut RACE libraries. cDNAs encoding both subunits had a consensus iron responsive element (IRE) in a conserved cap-distal location of their 5' UTR. An integrin-binding RGD motif found in the G subunit and conserved in Manduca may facilitate iron uptake through a calreticulin (mobilferrin)/integrin pathway. Calpodes and other insect ferritins have conserved cysteine residues to which fatty acids can be linked. Dynamic acylation of ferritin may slow but not prevent its passage out of the ER.