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Tetramerization domain mutations in KCNA5 Identified in pulmonary arterial hypertension patients affect channel kinetics and cause abnormal subcellular localization


Voltage-gated K⁺ (Kv) channels regulate resting membrane potential, apoptosis and proliferation in pulmonary artery smooth muscle cells (PASMC). The T1 domain of Kv\[alpha\]; subunits is necessary for channel assembly, subcellular localization and association with regulatory Kv\[beta\] subunits. As Kv channel dysfunction, including decreased Kv1.5 channel transcript, has been linked to idiopathic pulmonary arterial hypertension (IPAH), a genetically heterogeneous disease, the KCNA5 gene (which encodes Kv1.5) of IPAH patients was screened to identify IPAH- related mutations. Two non-synonymous polymorphisms were found in KCNA5 in IPAH patients. These encode a glycine to arginine mutation at position 182 (G182R) and a glutamate to aspartate mutation at position 211 (E211D), and localize to highly conserved residues within the T1 domain. To study the properties of the mutations, HEK-293 and COS-1 cells were transiently transfected with wildtype Kv1.5 (WT-Kv1.5), G182R, E211D or G182R/E211D channels. Whole-cell patch clamp recordings revealed that while the mutant subunits form functional channels, the G182R channel inactivates faster than the WT-Kv1.5 channel at potentials greater than -20 mV, while both the E211D and G182R/E211D channels inactivate faster than WT-Kv1.5 at potentials less than -20 mV. Furthermore, both activation and channel closure are slowed in mutant channels. Kv1.5 channels undergo complex glycosylation prior to cell surface expression. Immunoblot studies in HEK-293 cells suggest that a greater proportion of the G182R and G182R/ E211D channels are present in their immature form than WT- Kv1.5 channel. Furthermore, protein expression of the mutants is decreased in HEK-293 and COS-1 (G182R < 20% of WT-Kv1.5). Immunocytochemical analysis of HEK-293, COS-1 and PASMC confirm that while WT-Kv1.5 is robustly expressed on the cell surface, G182R, E211D and G182R/ E211D are retained intracellularly. While mutant channels maintain their ability to interact with Kv\[beta\] subunits, overexpression of Kv\[beta\]1.3 does not rescue G182R expression in COS-1 cells. Overall, the mutant channels inactivate more quickly and are localized to the plasma membrane less efficiently than the wildtype channel. These data suggest a role for the G182 and E211 residues in Kv1.5 channel kinetics and subcellular localization and suggest that, in a small subset of IPAH patients, these mutations may contribute to Kv1.5 channel dysfunction.

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