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. 1987 Jan;84(2):585–589. doi: 10.1073/pnas.84.2.585

Identification, purification, and functional reconstitution of the cyclic GMP-dependent channel from rod photoreceptors.

N J Cook, W Hanke, U B Kaupp
PMCID: PMC304255  PMID: 2432613

Abstract

The cyclic GMP-dependent cation channel from bovine rod outer segments has been purified to greater than 90% homogeneity by a rapid two-step chromatographic procedure. The purified channel has an apparent molecular mass of 63 kDa as determined by NaDodSO4/gel electrophoresis. When incorporated into the membrane of liposomes, the purified protein mediates the cyclic GMP-dependent efflux of entrapped Ca2+. The reconstituted channel protein exhibits properties similar to the cyclic GMP-dependent channel observed in excised patches of the plasma membrane and in disk membranes. Cyclic GMP activated the channel cooperatively (Hill coefficient n = 3.1) with an apparent Michaelis constant of approximately 11 microM. After reconstitution of the purified protein into a planar lipid bilayer, we recorded cyclic GMP-stimulated single-channel activity. The single-channel conductance at physiological salt concentrations and in the absence of divalent cations was 26 pS. The drug l-cis-diltiazem, shown to block the cyclic GMP-dependent channel in excised patches of the plasma membrane and in isolated disks of rod outer segments, was ineffective against the purified channel.

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Selected References

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