Abstract
We investigated conformational changes occurring in the C-linker and cyclic nucleotide-binding (CNB) domain of CNGA1 channels by analyzing the inhibition induced by thiol-specific reagents in mutant channels Q409C and A414C in the open and closed state. Cd2+ (200 μM) inhibited irreversibly mutant channels Q409C and A414C in the closed but not in the open state. Cd2+ inhibition was abolished in the mutant A414Ccys-free, in the double mutant A414C + C505T and in the tandem construct A414C + C505T/CNGA1, but it was present in the construct A414C + C505cys-free. The cross-linker reagent M-2-M inhibited mutant channel Q409C in the open state. M-2-M inhibition in the open state was abolished in the double mutant Q409C + C505T and in the tandem construct Q409C + C505T/CNGA1. These results show that Cα of C505 in the closed state is located at a distance between 4 and 10.5 Å from the Cα of A414 of the same subunit, but in the open state C505 moves towards Q409 of the same subunit at a distance that ranges from 10.5 to 12.3 Å from Cα of this residue. These results are not consistent with a 3-D structure of the CNGA1 channel homologous to the structure of HCN2 channels either in the open or in the closed state.
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Abbreviations
- CNG:
-
Cyclic nucleotide-gated
- CNBD:
-
Cyclic nucleotide-binding domain
- CSM:
-
Cysteine scanning mutagenesis
- MTS:
-
Methanethiosulfonate
- M-2-M:
-
1,2-Ethanediyl bismethanethiosulfonate
- M-4-M:
-
1,4-Butanediyl bismethanethiosulfonate
- DTT:
-
Dithiothreitol
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Acknowledgments
We thank Vincent Torre for the continuous support and very useful comments and suggestions and Anita Zimmerman for comments on the manuscript. We would like to thank Dr. Jessica Franzot for technical assistance and discussion in performing Western blot analysis. This work was supported by a HFSP grant, a COFIN grant (2006) from the Italian Ministry, a grant from CIPE (GRAND FVG) and a FIRB grant (RBLA03AF28_007) from MIUR.
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Nair, A.V., Anselmi, C. & Mazzolini, M. Movements of native C505 during channel gating in CNGA1 channels. Eur Biophys J 38, 465–478 (2009). https://doi.org/10.1007/s00249-008-0396-7
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DOI: https://doi.org/10.1007/s00249-008-0396-7