Biophys J, December 2002, p. 3283-3295, Vol. 83, No. 6

Movement of the C-Helix during the Gating of Cyclic Nucleotide-Gated Channels

INFM Section and International School for Advanced Studies, I-34014 Trieste, Italy

Movements within the cyclic nucleotide-binding domain of cyclic nucleotide-gated channels are thought to underlie the initial phase of channel gating (Tibbs, G. R., D. T. Liu, B. G. Leypold, and S. A. Siegelbaum. 1998. J. Biol. Chem. 273:4497-4505; Zong, X., H. Zucker, F. Hofmann, and M. Biel. 1998. EMBO J. 17:353-362; Matulef, K., G. E. Flynn, and W. N. Zagotta. 1999. Neuron. 24:443-452; Paoletti, P., E. C. Young, and S. A. Siegelbaum. 1999. J. Gen. Physiol. 113:17-33; Johnson, J. P., and W. N. Zagotta. 2001. Nature. 412:917-921). To investigate these movements, cysteine mutation was performed on each of the 28 residues (Leu-583 to Asn-610), which span the agonist-binding domain of the -subunit of the bovine rod cyclic nucleotide-gated channel. The effects of Cd²⁺ ions, 2-trimethylammonioethylmethane thiosulfonate (MTSET) and copper phenanthroline (CuP) on channel activity were examined, in excised inside-out patches in the presence and in the absence of a saturating concentration of cGMP. The application of 100 µM Cd²⁺ in the presence of saturating concentration of cGMP caused an irreversible and almost complete reduction of the current in mutant channels E594C, I600C, and L601C. In the absence of cGMP, the presence of 100 µM Cd²⁺ caused a strong current reduction in all cysteine mutants from Asp-588 to Leu-607, with the exception of mutant channels A589C, M592C, M602C, K603C, and L606C. The selective effect of Cd²⁺ ions was very similar to that observed when adding the oxidizing agent CuP to the bath medium, except for mutant channel G597C, where CuP caused a stronger current decrease (67 ± 7%) than Cd²⁺ (23 ± 4%). In the absence of cGMP, MTSET caused a reduction of the current by >40% in mutant channels L607C, L601C, I600C, G597C, and E594C, whereas in the presence of cGMP only mutant channel L601C was affected. The application of MTSET protected many mutant channels from the effects of Cd²⁺ and CuP. These results suggest that, when CNG channels are in the open state, residues from Asp-588 to Leu-607 are in an -helical structure, homologous to the C-helix of the catabolite gene activator protein (Weber, I. T., and T. A. Steitz. 1987. J. Mol. Biol. 198:311-326). Furthermore, residues Glu-594, Gly-597, Ile-600, and Leu-601 of these helices belonging to two different subunits must be in close proximity. In the closed state the C-helices are in a different configuration and undergo significant fluctuations.

Biophys J, December 2002, p. 3283-3295, Vol. 83, No. 6
© 2002 by the Biophysical Society   0006-3495/02/12/3283/13  $2.00

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