Child Heath Research Vacation Studentship - Summer 2015
Student: Jarka Naser
Supervisor(s): Dr Azizun Nessa and Professor Khalid Hussain
Title of Project: Rescuing defective KATP channels using novel molecular chaperones
Aim of Project: To optimise a system for analysing the membrane expression of KATP channels,
which are normally retained in the endoplasmic reticulum in pancreatic β-cells.
Brief description of project and results. Please also comment on the value of your
experience:
Congenital hyperinsulinism (CHI) is a rare heterogeneous disease presented by unregulated insulin
secretion, affecting approximately 1/50,000 newborns. Severe hypoglycaemia and permanent
neurological damage can result if patients are not treated and some may have to undergo a
pancreatectomy (surgical removal of the pancreas). One cause of CHI is due to mutations in the
ABCC8 and KCNJ11 genes which encode KATP channels that are expressed on the membrane of
pancreatic β-cells and have roles in insulin secretion. A subset of mutations can lead to defective
trafficking of KATP channels as they remain in the endoplasmic reticulum (ER).
Throughout the duration of this project, HEK293 cells were grown in T75 flasks until they were 80-90%
confluent. The cells were then split and seeded into 6 well plates. After 24 hours the cells were
transfected with different combinations of pDsRed2-ER (ER marker), Kir6.2-GFP (Kir6.2 protein
marker) and wild-type ABCC8/mutant ABCC8. A day after transfection the media was changed, and 24
hours later the cells were ready for confocal imaging. The fluorescence of GFP indicated the location of
the Kir6.2 protein when expressed in the cell, hence indicating the final location of KATP channels. The
confocal microscope was used to gather data on the trafficking analysis of KATP channels. In absence of
SUR1 (protein which co-assembles with Kir6.2 into KATP channels) coexpression, Kir6.2 was visually
found resident in the ER in some cells, whilst in its presence, it was found at the periphery.
Colocalisation was calculated using the Imaris software. A system has now been optimised in which
the membrane expression of KATP channel trafficking defects can be further studied: understanding the
molecular basis of trafficking defects is key for developing therapies which may avoid near-total
pancreatectomies. Therefore novel molecular chaperones such as Ivacaftor, a potentiator drug which
improves channel gating and corrects the G551D-CFTR mutation (in cystic fibrosis), can be tested
using the same protocol to see whether it enhances the membrane expression of KATP channels.
This scholarship has been an extremely beneficial learning experience. I understood the investigation
process of how research is coordinated by obtaining and analysing data. I had the opportunity to
experience kinaesthetic laboratory application of the academic theory I learned about CHI. This helped
to enhance my knowledge as I used all learning methods. As there is a large transition between
undergraduate and professional research, I gained invaluable insight into what it is like to research in
an academic environment, including the level of competency required. During this studentship I was
encouraged to work independently, and this has confirmed my interest in pursuing a research career. I
conducted imperative techniques such as tissue culture, DNA transfection, PCRs and gel
electrophoresis, as well as observing cloning and extraction of plasmid DNA from bacterial colonies.
The skills that I have gained would not have been developed as constructively through undergraduate
practical classes alone. Both my practical laboratory skills and my ability to comprehend and
synthesise information from peer-reviewed journals have been enhanced. I very much
enjoyed the working style, challenges, and laboratory atmosphere itself.