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K. E. Smith, S. E. Wilkie, M. Stocker, D. M. Hunt; Cone Dystrophy With Supernormal Electroretinogram (CDSE): Functional Analysis of Mutations in KCNV2 Encoding the Voltage-Gated Potassium Channel Subunit, Kv8.2. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2586.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the functional effect of pathogenic mutations in KCNV2 found in patients with CDSE. KCNV2 encodes a voltage-gated potassium (Kv) channel subunit, Kv8.2, known to form heteromeric channels with Kv2.1 subunits.
The coding region of Kv8.2 was cloned into various expression vectors and mutations introduced using site-directed mutagenesis. Confocal microscopy was used to determine the localisation of WT and mutant Kv8.2 subunits in COS7L cells on coexpression with the partner subunit, Kv2.1. The yeast two-hybrid system was used to determine which Kv channel subunits interact with WT Kv8.2 and to study the effect of mutations in Kv8.2 on its interaction with Kv2.1. Whole cell electrophysiological recordings were performed on transfected HEK 293 cells to determine the effects of the mutations on channel function.
When expressed in mammalian cells, Kv8.2 subunits were retained in the cell cytoplasm, but when coexpressed with Kv2.1 they colocalised with Kv2.1 at the plasma membrane. Whole cell recordings from cells expressing WT Kv8.2 showed a suppression of the Kv2.1 current amplitude and reduced inactivation properties of the channel. The L126Q and W188C substitutions occur at the intracellular N-terminus within or in close proximity to a T1 domain; yeast two hybrid experiments showed that the T1 domains of Kv8.2 subunits with either of these mutations were unable to interact with the T1 domain of Kv2.1. Whole cell recordings were consistent with this finding. Several of the mutations studied occur in the pore region; confocal imaging and electrophysiological experiments indicated that these mutant subunits were incorporated into channels with Kv2.1 subunits but that the resulting channels were non-functional.
For both types of mutations, the ability of the mutant Kv8.2 subunit to interact with Kv2.1 and/or to form functional channels was abolished, consistent with the concept that it is the absence of functional Kv8.2/2.1 channels which results in CDSE.
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