UBC Theses and Dissertations
The role of the T1 domain in the trafficking of Kv1.5 Jochova, Martina
The TI domain of Kv channels is important in the subfamily-specific assembly by clustering individual monomers to increase their chances of interaction into tetramers. Furthermore, TI domain plays roles in channel gating, stability as well as an interaction site with auxiliary subunits that ultimately modulate the function and expression of the channel. We identified a 10 amino acid region at the start of the TI domain that is an important prerequisite for the functional expression of Kv1.5 ion channels. A number of N-terminal and TI domain deletions were made that changed the ability of the channel to traffick to the cell surface. N-terminal deletions up to the TI domain (ΔN1 19 Kv1.5) and larger deletions of at least 60% of the TI domain (ΔN189 and ΔN209 Kv1.5) yielded functional Kv1.5 channels. However, smaller deletions of 15 (ΔN135 Kv1.5) and 43 (ΔN163 Kv1.5) amino acids of the TI domain yielded non-trafficking and non-functional Kv1.5 channels. These mutants formed aggregates and were localized in the ER and the degradation pathway. However, re-addition of the first 10 amino acids of the TI domain to the non-trafficking mutants rescued their trafficking to the plasma membrane. Furthermore, point mutations of residues thought to be involved in the intermolecular interaction during tetramer formation result in subtle alterations of channel trafficking ability. As well, point mutations of previously identified ER retention motifs (RXR) rescue the aggregation of the non-trafficking mutants with a very small rescue in their current density. We concluded that protein aggregation may be caused by number of states, such as exposure of ER retention motifs, disruption of important P-sheet secondary structures, disruption of tetramerization contacts, or exposure of large hydrophobic residues. Additionally, the trafficking and function of ΔN1 19, ΔN189, and ΔN209 Kv1.5 mutants may be affected by deletion of important sites involved in the modulation of Kv1.5 function, such as phosphorylation and interaction with auxiliary subunits. It is also possible that the combination of these individual processes affect the overall Kv1.5 cell surface expression.
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