Fluo 4-AM and Confocal Microscope to Measure JP45 Protein Interaction with RyR1 By Calcium Release in Co-Transfected HEK293 Cells

Fluo 4-AM and confocal microscope to measure JP45 protein interaction with RyR1 and calcium release in co-transfected HEK293 cells

Mohammed Althobiti¹, Rawdah Khoja², Dorota Fiszer³,Phil Hopkins³

1 King Fahad Security College, Riyadh, KSA

2 Ministry of Health, Alamal Mental Health Complex, Medina, KSA

3 Leeds Institute of Molecular Medicine, St James’s University Hospital, Leeds, UK

Introduction

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle. MH results from anaesthetics induced breakdown of calcium homeostasis. We have previously sequenced jsrp1, encoding JP-45, and demonstrated an increased frequency of the variant p.Gly150Ala (c.449G>C) in susceptable individuals (MHS) compared to normal controls (MHN).

Objectives

To explore the potential role of p.Gly150Ala in the aetiology of MH, the functional effect of mutant JP45 containing this variant or another newly identified variant p.Pro108Leu, was compared with wild type in HEK293 cells co-transfected with RYR1.

Materials and Methods

Firstly and by using the method of site-directed mutagenesis, we inserted the previously identified variants p.Gly150Ala (c.449G>C) and p.Pro108Leu in cDNA of the coding sequence of the JSRP1. Then we cloned the mutant JSRP1 cDNA for each variant in pCMV vector. We then used these cDNAs to transfect previously stably RYR1-transfected HEK293 cells, transiently with pCMV vector containing the wild type or mutant JSRP1. Finally, we have used the transfected cells to carry out calcium release measurements by using Fluo 4-AM and a confocal microscope to investigate the effects of these variants on the calcium release induced by caffeine.

Results

The preliminary results of calcium release measurements indicate a significant increase in the calcium release rate in the HEK293 cells expressing mutant JP45 compared with cells expressing the wild type JP45 or RyR1 only.

Conclusions

These preliminary results lend further support to RyR1-JP-45 interaction and a potential modifier role of the JP-45 protein in MH susceptibility.

Keywords: JSRP1, malignant hyperthermia, calcium release.