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

Student thesis: Master's ThesisMaster of Science by Research (MScR)


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 Award19 Mar 2024
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorStephen C Harmer (Supervisor) & Andrew F James (Supervisor)

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