title: Developmental regulation of the A-type potassium-channel current in hippocampal neurons: role of the Kvbeta 1.1 subunit.
abstract: The rapidly inactivating A-type K+ current (IA) is prominent in hippocampal neurons; and the speed of its inactivation may regulate electrical excitability. The auxiliary K+ channel subunit Kvbeta 1.1 confers fast inactivation to Shaker-related channels and is postulated to affect IA. Whole-cell patch clamp recordings of rat hippocampal pyramidal neurons in primary culture showed a developmental decrease in the time constant of inactivation (tau(in)) of voltage-gated K+ currents: 17.9+/-1.5 ms in young neurons (5-7 days in vitro; n=53, mean+/-S.E.M.); 9.9+/-1.0 ms in mature neurons (12-15 days in vitro; n=72, mean+/-S.E.M., P<0.01). During the same developmental time, the level of Kvbeta 1.1 transcript increased more than two-fold in vitro and in vivo, determined by semi-quantitative reverse transcriptase-polymerase chain reaction for hippocampus. The hypothesis that up-regulation of Kvbeta 1.1 led to the changes in tau(in) was tested in vitro, using antisense knockdown. Kvbeta 1.1-specific antisense DNA was introduced with a modified herpes virus co-expressing enhanced green fluorescent protein and knockdown of Kvbeta 1.1 was verified by immunocytochemistry. Following transduction with the antisense virus, mature neurons reverted to tau(in) values characteristic of young neurons: 18.3+/-2.4 ms (n=20). The effect of antisense knockdown on electrical excitability was tested using current-clamp protocols to induce repetitive firing. Treatment with the antisense virus increased the interspike interval over a range of membrane depolarization (baseline membrane potential=-40 to +20 mV). This effect was most pronounced at -40 mV, where the ISI of the first pair of action potentials was nearly doubled. These data indicate that Kvbeta 1.1 contributes to the developmental control of IA in hippocampal neurons and that the magnitude of effect is sufficient to regulate electrical excitability. Viral-mediated antisense knockdown of Kvbeta 1.1 is capable of decreasing the electrical excitability of post-mitotic hippocampal neurons, suggesting this approach has applicability to gene therapy of neurological diseases associated with hyperexcitability.
authors.affiliation: Department of Physiology, The University of Arizona, 1501 North Campbell Avenue, Tucson, AZ 85724, USA.
authors.forename: T
authors.initials: T
authors.lastname: Falk
authors.forename: R K
authors.initials: RK
authors.lastname: Kilani
authors.forename: L A
authors.initials: LA
authors.lastname: Strazdas
authors.forename: R S
authors.initials: RS
authors.lastname: Borders
authors.forename: J V
authors.initials: JV
authors.lastname: Steidl
authors.forename: A J
authors.initials: AJ
authors.lastname: Yool
authors.forename: S J
authors.initials: SJ
authors.lastname: Sherman
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002452:Cell Count; D002478:Cells, Cultured; D021983:Cyclophilins / Q000235:genetics / Q000378:metabolism; D016373:DNA, Antisense / Q000378:metabolism; D004553:Electric Conductivity; D005260:Female; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins; D018259:Herpesvirus 1, Human / Q000378:metabolism; D006624:Hippocampus / Q000196:embryology / Q000254:growth & development / Q000378:metabolism* / Q000821:virology; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D051662:Kv1.1 Potassium Channel; D051663:Kv1.2 Potassium Channel; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D009474:Neurons / Q000145:classification / Q000378:metabolism* / Q000821:virology; D018408:Patch-Clamp Techniques / Q000379:methods; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011247:Pregnancy; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D014162:Transfection / Q000379:methods
chemical_list: D016373:DNA, Antisense; C496972:Kcna2 protein, rat; D051663:Kv1.2 Potassium Channel; D008164:Luminescent Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D012333:RNA, Messenger; D051662:Kv1.1 Potassium Channel; D049452:Green Fluorescent Proteins; D021983:Cyclophilins