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.

Structure and alternative splicing of the gene encoding alpha1G, a human brain T calcium channel alpha1 subunit.

The structure of CACNA1G, the gene encoding alpha1G, 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 38 exons, two of them newly-identified, spanning at least 66490 basepairs of chromosome 17q22. Alternative splicing of the RNA occurs at six sites: cassette exons 14, 26, 34 and 35, an internal donor in exon 25 and protein-coding intron 38B. Additionally, the RNA can be polyadenylated at either of two sites. Alternative splicing of CACNA1G RNA may lead to expression of as many as 24 distinct protein products, ranging from 2171 to 2377 amino-acids residues.

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.

Mechanism of modulation of the voltage-gated skeletal and cardiac muscle sodium channels by fatty acids.

Voltage-gated rat skeletal muscle and cardiac Na+ channels are modulated by exogenous unsaturated fatty acids. Application of 1-10 microM arachidonic or oleic acids reversibly depressed Na+ channel conductance and shifted the inactivation curve to hyperpolarizing potentials. These effects were not prevented by inhibitors of lipoxygenase, cyclooxygenase, cytochrome P-450 epoxygenase, or protein kinase C. Neither palmitic acid nor methyl ester oleate had an effect on the inward Na+ current, suggesting that trivial variations in membrane fluidity are not responsible for the Na+ current depression or kinetic changes. Arachidonic acid altered fast Na+ inactivation without changing the slow inactivation kinetics. Moreover, skeletal muscle Na+ channel gating currents were markedly decreased by 2 microM arachidonic acid. Finally, nonstationary noise analysis indicated that both the number of channels and the open probability were slightly decreased without change in the single-channel conductance. These data suggest that unsaturated fatty acids such as arachidonic and oleic acids 1) specifically regulate voltage-gated Na+ channels and 2) interact directly with Na+ channels, perhaps at a fatty acid binding domain, by decreasing the total gating charge and altering fast-inactivation kinetics.

The type 1 inositol 1,4,5-trisphosphate receptor gene is altered in the opisthotonos mouse.

The opisthotonos (opt) mutation arose spontaneously in a C57BL/Ks-db2J colony and is the only known, naturally occurring allele of opt. This mutant mouse was first identified based on its ataxic and convulsive phenotype. Genetic and molecular data presented here demonstrate that the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) protein, which serves as an IP3-gated channel to release calcium from intracellular stores, is altered in the opt mutant. A genomic deletion in the IP3R1 gene removes two exons from the IP3R1 mRNA but does not interrupt the translational reading frame. The altered protein is predicted to have lost several modulatory sites and is present at markedly reduced levels in opt homozygotes. Nonetheless, a strong calcium release from intracellular stores can be elicited in cerebellar Purkinje neurons treated with the metabotropic glutamate receptor (mGluR) agonist quisqualate (QA). QA activates Group 1 mGluRs linked to GTP-binding proteins that stimulate phospholipase C and subsequent production of the intracellular messenger IP3, leading to calcium mobilization via the IP3R1 protein. The calcium response in opt homozygotes shows less attenuation to repeated QA application than in control littermates. These data suggest that the convulsions and ataxia observed in opt mice may be caused by the physiological dysregulation of a functional IP3R1 protein.

Enhancement of the Shaker B delta6-46 current by fatty acids depends on the activation of the lipoxygenase metabolic pathway.

Voltage-gated K+ channels are modulated by extracellular free unsaturated fatty acids. Both increases and attenuations of K+ channels activities have been observed. We studied the effect of cis-unsaturated fatty acids on Shaker B delta6-46 and the endogenous outward rectifier K+ channels expressed in COS cell line, using the whole cell recording technique. For both K+ channels, exogenously applied unsaturated fatty acids dramatically increased the outward K+ currents. This enhancement was not mediated by cylooxygenase or epoxygenase (P450) enzymes. However, nordihydroguaiaretic acid, a lipoxygenase metabolic pathway blocker, did prevent the arachidonic acid-induced Shaker current enhancement.

Serine-1321-independent regulation of the mu 1 adult skeletal muscle Na+ channel by protein kinase C.

The adult skeletal muscle Na+ channel mu1 possesses a highly conserved segment between subunit domains III and IV containing a consensus protein kinase C (PKC) phosphorylation site that, in the neuronal isoform, acts as a master control for "convergent" regulation by PKC and cAMP-dependent protein kinase. It lacks an approximately 200-aa segment between domains I and II though to modulate channel gating. We here demonstrate that mu1 is regulated by PKC (but not cAMP-dependent protein kinase) in a manner distinct from that observed for the neuronal isoforms, suggesting that under the same conditions muscle excitation could be uncoupled from motor neuron input. Maximal phosphorylation by PKC, in the presence of phosphatase inhibitors, reduced peak Na+ currents by approximately 90% by decreasing the maximal conductance, caused a -15 mV shift in the midpoint of steady-state inactivation, and caused a slight speeding of inactivation. Surprisingly, these effects were not affected by mutation of the conserved serine (serine-1321) in the interdomain III-IV loop. the pattern of current suppression and gating modification by PKC resembles the response of muscle Na+ channels to inhibitory factors present in the serum and cerebrospinal fluid of patients with Guillain-Barré syndrome, multiple sclerosis, and idiopathic demyelinating polyradiculoneuritis.

The microI skeletal muscle sodium channel: mutation E403Q eliminates sensitivity to tetrodotoxin but not to mu-conotoxins GIIIA and GIIIB.

Voltage-sensitive Na channels from nerve and muscle are blocked by the guanidinium toxins tetrodotoxin (TTX) and saxitoxin (STX). Mutagenesis studies of brain RII channels have shown that glutamate 387 (E387) is essential for current block by these toxins. We demonstrate here that mutation of glutamate 403 (E403) of the adult skeletal muscle microI channel (corresponding to E387 of RII) also prevents current blockade by TTX and STX, and by neo-saxitoxin. However, the mutation fails to prevent blockade by the peptide neurotoxins, mu-conotoxin GIIIA and GIIIB; these toxins are thought to bind to the same or overlapping sites with TTX and STX. The E403Q mutation may have utility as a marker for exogenous Na channels in transgenic expression studies, since there are no known native channels with the same pharmacological profile.

A glial-specific voltage-sensitive Na channel gene maps close to clustered genes for neuronal isoforms on mouse chromosome 2.

A variety of glial cell types express saxitoxin (STX)-binding voltage-sensitive Na channels (1,2), although the possible role of impulse conduction in these cells is not understood. Gautron et al. (1992) recently identified a 7.5 kb species of mRNA in type 1 astrocytes cultured from rat brain cerebrum that hybridized with a "common" Na channel probe but not with brain isoform-specific cDNA probes. Sequence data from cloned cDNAs demonstrate that it encodes a structurally atypical Na channel isoform. We have prepared a cDNA probe specific for a portion of subunit domain IV of the glial channel and mapped the location of the corresponding gene (Scn7a) to mouse chromosome 2. The Scn7a gene mapped 0.9 (+/- 0.9) cM distal to the Gcg locus; the location of the corresponding human gene (SCN7A) is predicted to be in the q36-q37 region of chromosome 2. This site lies just outside a cluster of genes for the brain-specific Na channel isoforms RI, RII and RIII which map proximal to Gcg (17). The presence of at least four genes from two distinct Na channel subfamilies suggests that multiple genetic defects for central and peripheral nervous system disorders ultimately may be linked to this area.

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.

A single B1 subunit mapped to mouse chromosome 7 may be a common component of Na channel isoforms from brain, skeletal muscle and heart.

A beta 1 subunit associated with one or more isoforms of brain voltage-sensitive Na channels has previously been cloned, sequenced and expressed. Northern and Western blot analyses have suggested that homologues to this protein are expressed in skeletal muscle and heart. Here, reverse transcriptase-polymerase chain reaction (RT-PCR)/cloning reveals that transcripts encoding identical beta 1 subunit ORFs are expressed in adult rat brain, skeletal muscle and heart. Heterologous co-expression of beta 1 with brain (RIIA) and skeletal muscle (mu 1) alpha subunits caused a stabilization of normal, rapidly inactivating (mode 1) gating relative to anomalous, non-inactivating (mode 2) states and a negative shift in steady state inactivation. Chromosome mapping of the beta 1 subunit showed a single locus (Scn1b) in mouse chromosome 7 1.8 cM (+/- 1.2 cM) distal to D19F11S1h and 0.9 cM (+/- 0.9 cM) proximal to Pkca. This locus is in the region of the mouse mutant "quivering," characterized by a variety of neurological disorders and muscle paralysis. A mutation in a single beta 1 subunit forming functional complexes with multiple Na channel isoforms could underlie these deficits.

Functional consequences of a Na+ channel mutation causing hyperkalemic periodic paralysis.

Hyperkalemic periodic paralysis (HYPP), one of several inheritable myotonic diseases, results from genetic defects in the human skeletal muscle Na+ channel. In some pedigrees, HYPP is correlated with a single base pair substitution resulting in a Met replacing Thr704 in the fifth transmembrane segment of the second domain. This region is totally conserved between the human and rat channels. We have introduced the human mutation into the corresponding region of the rat muscle Na+ channel cDNA and expressed it in human embryonic kidney 293 cells. Patch-clamp recordings show that this mutation shifts the voltage dependence of activation by 10-15 mV in the negative direction. The shift results in a persistent Na+ current that activates near -70 mV; this phenomenon could underlie the abnormal muscle activity observed in patients with HYPP.

muI Na+ channels expressed transiently in human embryonic kidney cells: biochemical and biophysical properties.

We describe the transient expression of the rat skeletal muscle muI Na+ channel in human embryonic kidney (HEK 293) cells. Functional channels appear at a density of approximately 30 in a 10 microns 2 patch, comparable to those of native excitable cells. Unlike muI currents in oocytes, inactivation gating is predominantly (approximately 97%) fast, although clear evidence is provided for noninactivating gating modes, which have been linked to anomalous behavior in the inherited disorder hyperkalemic periodic paralysis. Sequence-specific antibodies detect a approximately 230 kd glycopeptide. The majority of molecules acquire only neutral oligosaccharides and are retained within the cell. Electrophoretic mobility on SDS gels suggests the molecules may acquire covalently attached lipid. The channel is readily phosphorylated by activation of the protein kinase A and protein kinase C second messenger pathways.

Multiple gating modes and the effect of modulating factors on the microI sodium channel.

Macroscopic current from the microI skeletal muscle sodium channel expressed in Xenopus oocytes shows inactivation with two exponential components. The major, slower component's amplitude decreases with rapid pulsing. When microI cRNA is coinjected with rat skeletal muscle or brain mRNA the faster component becomes predominant. Individual microI channels switch between two principal gating modes, opening either only once per depolarization, or repeatedly in long bursts. These two modes differ in both activation and inactivation kinetics. There is also evidence for additional gating modes. It appears that the equilibrium among gating modes is influenced by a modulating factor encoded in rat skeletal muscle and brain mRNA. The modal gating is similar to that observed in hyperkalemic periodic paralysis.

Paramyotonia congenita and hyperkalemic periodic paralysis map to the same sodium-channel gene locus.

Paramyotonia congenita (PC), an autosomal dominant muscle disease, shares some clinical and electrophysiological similarities with another myotonic muscle disorder, hyperkalemic periodic paralysis (HYPP). However, clinical and electrophysiologic differences allow differentiation of the two disorders. The HYPP locus was recently shown to be linked to a skeletal muscle sodium-channel gene probe. We now report that PC maps to the same locus (LOD score 4.4, theta = 0 at assumed penetrance of .95). These linkage results, coupled with physiological data demonstrating abnormal sodium-channel function in patients with PC, implicate a sodium-channel gene as an important candidate for the site of mutation responsible for PC. Furthermore, this is strong evidence for the hypothesis that PC and HYPP are allelic disorders.

Voltage-sensitive Na+ channels: motifs, modes and modulation.

Much recent progress has been made in understanding the structural organization and functional properties of voltage-dependent Na+ channels, in particular in the areas of activation, ion conductance, and inactivation. At the same time, however, electrophysiological studies have revealed new, more complex functional properties in the form of at least two gating modes and the existence of as yet unidentified modulatory factors.

Analysis in a large hyperkalemic periodic paralysis pedigree supports tight linkage to a sodium channel locus.

Hyperkalemic periodic paralysis (HYPP) is an autosomal dominant muscle disease with electrophysiological abnormalities suggesting a defect in a voltage-gated sodium channel (NaCh) gene. A human NaCh gene was recently shown to cosegregate with the disease allele in a family with HYPP. Using an independent clone, we have demonstrated close genetic linkage between an NaCh gene and the HYPP locus in another family. With physiological data demonstrating abnormal NaCh function in HYPP patients, the absence of any obligate recombinations in the two families strengthens the argument that this NaCh gene is the site of the defect in this disorder.

A rapid ion-exchange assay for detergent-solubilized inositol 1,4,5-trisphosphate receptors.

We describe a rapid ion-exchange syringe assay for [3H]inositol 1,4,5-trisphosphate binding to detergent-solubilized receptors. In extracts of rat cerebellar membranes, the assay resolves rapidly dissociating ligand complexes, detecting two to three times higher receptor abundance than conventional gel filtration spun column assays, and provides evidence for two classes of IP3-binding sites, representing 0.5-1.0% of total cerebellar membrane protein. Receptors purified from bovine and rat cerebellum exhibit a single class of high-affinity sites, with equilibrium dissociation constants (Kd = 4-8 nM) reflecting 20 to 25-fold higher affinity than reported in studies with spun-column methods.