title: Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit.
abstract: An in vivo experimental strategy, involving cardiac-specific expression of a mutant Kv 2.1 subunit that functions as a dominant negative, was exploited in studies focused on exploring the role of members of the Kv2 subfamily of pore-forming (alpha) subunits in the generation of functional voltage-gated K(+) channels in the mammalian heart. A mutant Kv2.1 alpha subunit (Kv2.1N216) was designed to produce a truncated protein containing the intracellular N terminus, the S1 membrane-spanning domain, and a portion of the S1/S2 loop. The truncated Kv2.1N216 was epitope tagged at the C terminus with the 8-amino acid FLAG peptide to generate Kv2. 1N216FLAG. No ionic currents are detected on expression of Kv2. 1N216FLAG in HEK-293 cells, although coexpression of this construct with wild-type Kv2.1 markedly reduced the amplitudes of Kv2. 1-induced currents. Using the alpha-myosin heavy chain promoter to direct cardiac specific expression of the transgene, 2 lines of Kv2. 1N216FLAG-expressing transgenic mice were generated. Electrophysiological recordings from ventricular myocytes isolated from these animals revealed that I(K, slow) is selectively reduced. The attenuation of I(K, slow) is accompanied by marked action potential prolongation, and, occasionally, spontaneous triggered activity (apparently induced by early afterdepolarizations) is observed. The time constant of inactivation of I(K, slow) in Kv2. 1N216FLAG-expressing cells (mean+/-SEM=830+/-103 ms; n=17) is accelerated compared with the time constant of I(K, slow) inactivation (mean+/-SEM=1147+/-57 ms; n=25) in nontransgenic cells. In addition, unlike I(K, slow) in wild-type cells, the component of I(K, slow) remaining in the Kv2.1N216FLAG-expressing cells is insensitive to 25 mmol/L tetraethylammonium. Taken together, these observations suggest that there are 2 distinct components of I(K, slow) in mouse ventricular myocytes and that Kv2 alpha subunits underlie the more slowly inactivating, tetraethylammonium-sensitive component of I(K, slow). In vivo telemetric recordings also reveal marked QT prolongation, consistent with a defect in ventricular repolarization, in Kv2.1N216FLAG-expressing transgenic mice.
authors.affiliation: Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, MO 63110, USA.
authors.forename: H
authors.initials: H
authors.lastname: Xu
authors.forename: D M
authors.initials: DM
authors.lastname: Barry
authors.forename: H
authors.initials: H
authors.lastname: Li
authors.forename: S
authors.initials: S
authors.lastname: Brunet
authors.forename: W
authors.initials: W
authors.lastname: Guo
authors.forename: J M
authors.initials: JM
authors.lastname: Nerbonne
mesh_terms: D000200:Action Potentials; D000818:Animals; D002460:Cell Line; D051668:Delayed Rectifier Potassium Channels; D004553:Electric Conductivity; D004562:Electrocardiography; D004594:Electrophysiology; D005799:Genes, Dominant; D006352:Heart Ventricles; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D011188:Potassium / Q000502:physiology; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051669:Shab Potassium Channels; D013997:Time Factors
chemical_list: D051668:Delayed Rectifier Potassium Channels; C496988:KCNB1 protein, human; C496990:Kcnb1 protein, mouse; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D020033:Protein Isoforms; D051669:Shab Potassium Channels; D011188:Potassium