Investigating the effects of Long QT Syndrome associated Caveolin-3 variants on trafficking in HEK293 cells

Abstract

Congenital long QT syndrome (cLQTS) is a rare cardiac disorder that disrupts the normal
electrical activity of the heart. cLQTS was first linked to mutations in the genes that code for
ion channels; however, further studies discovered associations with mutations in proteins
that participate in the trafficking/function of ion channels. Mutations in the CAV3 gene,
which encodes the Caveolin-3 (Cav-3) protein, have been linked to cLQTS type 9 (LQT9).
Whether CAV3 mutations cause LQT9 by perturbing the trafficking of Cav-3 is unclear and
has not been examined in a quantitative manner.
Therefore, this project aimed to develop an imaging-based assay to quantitatively assess the
cellular localisation and trafficking status of two LQT9-associated Cav-3 variants (F97C and
S141R) and a Limb-girdle muscular dystrophy associated variant (A46T), which is reported to
be trafficking defective. The cellular localisation of untagged and meGFP-tagged wild-type
(WT)/variant Cav-3 was determined using immunostaining, confocal microscopy and colocalisation analysis in transfected Human Embryonic Kidney-293 cells.
The co-localisation analysis revealed that the cellular localisation profile of untagged F97C
and S141R was not significantly different from untagged WT Cav-3. In contrast, untagged
A46T Cav-3 showed reduced plasma membrane co-localisation and increased retention in
the endoplasmic reticulum (ER) compared to untagged WT Cav-3, suggesting a defect in
trafficking. In addition, the fusion of meGFP to the N-terminus of Cav-3 resulted in a
significantly higher degree of ER retention for both WT and variant Cav-3 compared to
untagged Cav-3.
This study is the first to quantitatively characterise the trafficking of LQT9-associated Cav-3
mutants. In brief, F97C and S141R were found not to perturb trafficking. However, Nterminus meGFP fusion of Cav-3 resulted in changes in trafficking, which has implications for
the use of similar constructs in the future.

Date of Award	19 Mar 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Stephen C Harmer (Supervisor) & Andrew F James (Supervisor)