Irreversible Activation of Cyclic Nucleotide-Gated Ion Channels by Sulfhydryl-Reactive Derivatives of Cyclic GMP

R. Lane Brown; Tammie L Haley; Sheila D Snow

doi:10.1021/bi9920735

First discovered in the sensory epithelium of the visual and olfactory systems, cyclic nucleotide-gated (CNG) ion channels have now been found in tissues throughout the body. Native rod CNG channels are tetramers composed of homologous, but distinct, α- and β-subunits. The goal of this study was to develop a novel method for targeting covalent attachment of cGMP to individual subunit types. Toward this goal, we have found that treatment of membrane patches expressing rod α-subunit channels with sulfhydryl-reactive derivatives of cGMP resulted in irreversible activation. The persistent currents were sensitive to block by both Mg2+ and tetracaine. Pretreatment of the patch with the sulfhydryl-blocking reagents N-ethylmaleimide (NEM) and bis-dithionitrobenzoic acid (DTNB) prevented covalent activation; the effect of DTNB was reversed by reduction with DTT. Furthermore, the process of covalent activation was dramatically slowed by the presence of an excess of 8-Br-cGMP. These results suggested that covalent activation resulted from the tethering of cGMP near the channel's ligand-binding sites by reaction with an endogenous cysteine. The α-subunit of the rod channel contains seven cysteine residues, and we set out to determine the site of attachment by site-directed mutagenesis. Surprisingly, irreversible activation was not abolished by elimination of all seven cysteine residues. This result suggests that the site of attachment is on a tightly associated protein, rather than on the channel protein itself. To further investigate these results, we treated patches containing irreversibly activated channels with 100 μg/mL trypsin and discovered two modes of covalent activation. One type developed rapidly and was removed by trypsin treatment, and the second developed slowly and was resistant to trypsin treatment. Both types of covalent activation were present in all mutants tested and were also present when CNG channels were expressed in HEK-293 cells. These results suggest that CNG channel subunits may associate with endogenous proteins when they are expressed in heterologous systems.

Irreversible Activation of Cyclic Nucleotide-Gated Ion Channels by Sulfhydryl-Reactive Derivatives of Cyclic GMP

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