RNA Editing Generates a Diverse Array of Transcripts Encoding Squid Kv2 K+ Channels with Altered Functional Properties

We have cloned a Kv2 potassium channel from squid optic lobe termed sqKv2. Multiple overlapping sqKv2 cDNA clones differed from one another at specific positions by purine transitions. To test whether the purine transitions were generated by RNA editing, we compared a 360 nucleotide genomic sequence with corresponding cDNA sequences (encoding S4-S6) isolated from individual animals and lying on a single gene and exon. cDNA sequences differed from genomic sequence at 17 positions, resulting in 28 unique sequences. There was invariantly an adenosine in the genomic sequence and a guanosine in the edited cDNA sequences. Two of the edits altered the rates of channel closure and slow inactivation. These results extend selective RNA editing to invertebrate taxa and represents a novel mechanism for the posttranscriptional modulation of voltage-gated ion channels.

[1]  S. Roberds,et al.  Developmental expression of cloned cardiac potassium channels , 1991, FEBS letters.

[2]  P. Seeburg,et al.  A standardized nomenclature for adenosine deaminases that act on RNA. , 1997, RNA.

[3]  H. Guy,et al.  Atomic scale structure and functional models of voltage-gated potassium channels. , 1992, Biophysical journal.

[4]  R. T. D'Aquila,et al.  Maximizing sensitivity and specificity of PCR by pre-amplification heating , 1991, Nucleic Acids Res..

[5]  F Bezanilla,et al.  Gating of Shaker K+ channels: I. Ionic and gating currents. , 1994, Biophysical journal.

[6]  B. Bass,et al.  Preferential selection of adenosines for modification by double‐stranded RNA adenosine deaminase. , 1994, The EMBO journal.

[7]  T Kuner,et al.  Control of kinetic properties of AMPA receptor channels by nuclear RNA editing. , 1994, Science.

[8]  A. Brown,et al.  Rescue of lethal subunits into functional K+ channels. , 1994, Biophysical journal.

[9]  S. Snyder,et al.  A novel K+ channel with unique localizations in mammalian brain: Molecular cloning and characterization , 1992, Neuron.

[10]  G. Freeman,et al.  Sequential expression of new gene programs in inducer T-cell clones. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[11]  P. Seeburg,et al.  RNA editing in brain controls a determinant of ion flow in glutamate-gated channels , 1991, Cell.

[12]  A. Hodgkin,et al.  A quantitative description of membrane current and its application to conduction and excitation in nerve , 1952, The Journal of physiology.

[13]  R. MacKinnon,et al.  The aromatic binding site for tetraethylammonium ion on potassium channels , 1992, Neuron.

[14]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.

[15]  R. MacKinnon,et al.  Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel , 1991, Science.

[16]  B. Rudy,et al.  A-type potassium channels expressed from Shaker locus cDNA. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Richard W. Aldrich,et al.  Two types of inactivation in Shaker K+ channels: Effects of alterations in the carboxy-terminal region , 1991, Neuron.

[18]  Albert Ribera,et al.  Xenopus spinal neurons express Kv2 potassium channel transcripts during embryonic development , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[19]  Roderick MacKinnon,et al.  Contribution of the S4 Segment to Gating Charge in the Shaker K+ Channel , 1996, Neuron.

[20]  F. Bezanilla,et al.  Activation of squid axon K+ channels. Ionic and gating current studies , 1985, The Journal of general physiology.

[21]  I. Llano,et al.  Ionic conductances of squid giant fiber lobe neurons , 1986, The Journal of general physiology.

[22]  Y. Jan,et al.  Four cDNA clones from the Shaker locus of Drosophila induce kinetically distinct A-type potassium currents in Xenopus oocytes , 1988, Neuron.

[23]  Francisco Bezanilla,et al.  Voltage-Sensing Residues in the S2 and S4 Segments of the Shaker K+ Channel , 1996, Neuron.

[24]  Y. Jan,et al.  Specification of subunit assembly by the hydrophilic amino-terminal domain of the Shaker potassium channel. , 1992, Science.

[25]  G. Ehrenstein,et al.  Slow changes of potassium permeability in the squid giant axon. , 1966, Biophysical journal.

[26]  R. Emeson,et al.  Regulation of serotonin-2C receptor G-protein coupling by RNA editing , 1997, Nature.

[27]  R. Emeson,et al.  Glutamate receptor RNA editing in vitro by enzymatic conversion of adenosine to inosine , 1995, Science.

[28]  J. Rosenthal,et al.  Molecular identification of SqKv1A. A candidate for the delayed rectifier K channel in squid giant axon , 1996, The Journal of general physiology.

[29]  Y. Jan,et al.  Multiple potassium–channel components are produced by alternative splicing at the Shaker locus in Drosophila , 1988, Nature.

[30]  L. Kaczmarek,et al.  A shab potassium channel contributes to action potential broadening in peptidergic neurons , 1994, Neuron.

[31]  P. Seeburg,et al.  Q/R site editing in kainate receptor GluR5 and GluR6 pre-mRNAs requires distant intronic sequences. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[32]  A. L. Goldin Expression of ion channels by injection of mRNA into Xenopus oocytes. , 1991, Methods in cell biology.

[33]  W. Rutter,et al.  Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. , 1979, Biochemistry.

[34]  F Bezanilla,et al.  Inactivation of the sodium channel. II. Gating current experiments , 1977, The Journal of general physiology.

[35]  L. Salkoff,et al.  A mouse brain homolog of the Drosophila Shab K+ channel with conserved delayed-rectifier properties , 1991, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[36]  F Bezanilla,et al.  Potassium conductance of the squid giant axon. Single-channel studies , 1988, The Journal of general physiology.

[37]  H. Sambrook Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[38]  T. Maniatis,et al.  Editing of glutamate receptor subunit B pre-mRNA in vitro by site-specific deamination of adenosine , 1995, Nature.

[39]  Alan L. Goldin,et al.  Topology of the Shaker Potassium Channel Probed with Hydrophilic Epitope Insertions , 1997, The Journal of cell biology.

[40]  P. Pfaffinger,et al.  Molecular recognition and assembly sequences involved in the subfamily-specific assembly of voltage-gated K+ channel subunit proteins , 1995, Neuron.

[41]  B. Sakmann,et al.  Determinants of ca2+ permeability in both TM1 and TM2 of high affinity kainate receptor channels: Diversity by RNA editing , 1993, Neuron.

[42]  Brenda L. Bass,et al.  RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase , 1996, Nature.

[43]  T. J. Baldwin,et al.  Characterization of a mammalian cDNA for an inactivating voltage-sensitive K+ channel , 1991, Neuron.

[44]  R. MacKinnon,et al.  Mutations affecting TEA blockade and ion permeation in voltage-activated K+ channels. , 1990, Science.

[45]  R. Aldrich,et al.  Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. , 1993, Receptors & channels.

[46]  A. VanDongen,et al.  A novel potassium channel with delayed rectifier properties isolated from rat brain by expression cloning , 1989, Nature.

[47]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[48]  K. A. Eckert,et al.  High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase , 1990, Nucleic Acids Res..

[49]  M. Kozak An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. , 1987, Nucleic acids research.

[50]  Yuichi Kanaoka,et al.  Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence , 1984, Nature.

[51]  R. MacKinnon Determination of the subunit stoichiometry of a voltage-activated potassium channel , 1991, Nature.

[52]  A. Brennicke,et al.  RNA editing. , 1999, FEMS microbiology reviews.

[53]  Xinghai Chen,et al.  Deletion analysis of K+ channel assembly , 1993, Neuron.

[54]  D J Baro,et al.  Shab gene expression in identified neurons of the pyloric network in the lobster stomatogastric ganglion. , 1994, Receptors & channels.

[55]  L. Salkoff,et al.  A family of putative potassium channel genes in Drosophila. , 1989, Science.

[56]  P. Seeburg,et al.  Editing of α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid Receptor GluR-B Pre-mRNA in Vitro Reveals Site-selective Adenosine to Inosine Conversion(*) , 1995, The Journal of Biological Chemistry.