Transfection of receptor subunits into HEK293 (HEK-293) cells using Magnetofection

Receptor subunits were transiently expressed in HEK293 (HEK-293) cells using the Magnetofection(TM) (CombiMag) method.

This paper demonstrated the high efficiency of Magnetofection(TM) from OZ Biosciences to transiently transfect cell lines for receptor subunits expression.

article reference: J Biol Chem. 2013 Oct 9.

New hyperekplexia mutations provide insight into glycine receptor assembly, trafficking and activation mechanisms.

Bode A, Wood SE, Mullins JG, Keramidas A, Cushion TD, Thomas RH, Pickrell WO, Drew CJ, Masri A, Jones EA, Vassallo G, Born AP, Alehan F, Aharoni S, Bannasch G, Bartsch M, Kara B, Krause A, Karam EG, Matta S, Jain V, Mandel H, Freilinger M, Graham GE, Hobson E, Chatfield S, Vincent-Delorme C, Rahme JE, Afawi Z, Berkovic SF, Howell OW, Vanbellinghen JF, Rees MI, Chung SK, Lynch JW.

Abstract

Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalised hypertonia that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels (pLGICs) with a definitive and clinically well-stratified linkage to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analysed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations of which nine were novel. Electrophysiological analysis demonstrated that the dominant mutations p.Q226E, p.V280M and p.R414H induced spontaneous channel activity indicating this is a recurring mechanism in hGlyR pathophysiology. p.Q226E, at the top of TM1, most likely induced tonic activation via an enhanced electrostatic attraction to p.R271 at the top of TM2, suggesting a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors, and represents a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A and p.E375X precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated into functional hGlyRs together with unmutated α1 or α1 plus β subunits. These aberrant receptors exhibit significantly reduced glycine sensitivity. To our knowledge, this is the first suggestion that subunits lacking TM4 domains might be incorporated into functional pLGIC receptors.

CombiMag from OZ Biosciences is a very efficient transfection reagent based on Magnetofection™ technology.