Recombinant human voltage-gated skeletal muscle sodium channels are pharmacologically functional in planar lipid bilayers.
Human voltage-gated sodium ion channels are major sites of action for drugs and toxins that modulate cellular excitability, and are therefore key molecular targets for ion channel research, high throughput screening for new drugs, and toxin detection. Protein suitable for these applications must be produced in a functionally active form. We report the successful use of ion metal affinity chromatography (IMAC) to purify C-terminal polyhistidine tagged human skeletal muscle voltage-gated sodium (hSkM1-HT) channels from Sf9 insect cells; hSkM1 channels were pharmacologically functional when reconstituted into liposomes and incorporated into planar bilayer lipid membranes. hSkM1-HT single channel currents activated by veratridine had a conductance of 21 pS and those activated by brevetoxin, 16 pS. Channel activity was inhibited by tetrodotoxin and saxitoxin. This protein is suitable for the development of biosensor and high throughput screening technologies.
Zhang
Yan Li
YL
AgResearch Limited, Grasslands Research Centre, Palmerston North, New Zealand. Yan-Li.Zhang@agreearch.co.nz
Dunlop
James
J
Dalziel
Julie E
JE
D015374:Biosensing Techniques / Q000379:methods; D006801:Humans; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000737:chemistry*; D008564:Membrane Potentials; D018482:Muscle, Skeletal / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D012530:Saxitoxin / Q000737:chemistry*; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics; D013779:Tetrodotoxin / Q000737:chemistry*
D008051:Lipid Bilayers; D011994:Recombinant Proteins; D015222:Sodium Channels; D012530:Saxitoxin; D013779:Tetrodotoxin