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A comparison of electrophysiological properties of the CNGA1, CNGA1tandem and CNGA1cys-free Channels

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Abstract

Three constructs are used for the analysis of biophysical properties of CNGA1 channels: the WT CNGA1 channel, a CNGA1 channel where all endogenous cysteines were removed (CNGA1cys-free) and a construct composed of two CNGA1 subunits connected by a small linker (CNGA1tandem). So far, it has been assumed, but not proven, that the molecular structure of these ionic channels is almost identical. The I/V relations, ionic selectivity to alkali monovalent cations, blockage by tetracaine and TMA+ were not significantly different. The cGMP dose response and blockage by TEA+ and Cd2+ were instead significantly different in CNGA1 and CNGA1cys-free channels, but not in CNGA1 and CNGA1tandem channels. Cd2+ blocked irreversibly the mutant channel A406C in the absence of cGMP. By contrast, Cd2+ did not block the mutant channel A406C in the CNGA1cys-free background (A406Ccys-free), but an irreversible and almost complete blockage was observed in the presence of the cross-linker M–4–M. Results obtained with different MTS cross-linkers and reagents suggest that the 3D structure of the CNGA1cys-free differs from that of the CNGA1 channel and that the distance between homologous residues at position 406 in CNGA1cys-free is longer than in the WT CNGA1 by several Angstroms.

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Abbreviations

CNG:

Cyclic nucleotide-gated

CNBD:

Cyclic nucleotide-binding domain

CSM:

Cysteine scanning mutagenesis

MTS:

Methanethiosulfonate

MTSET:

2-(Trimethylammonium)ethyl] methanethiosulfonate bromide

MTSPT:

3-(Trimethylammonium)propyl methanethiosulfonate bromide

MTS-PtrEA:

3-(Triethylammonium)propyl methanthiosulfonate bromide

M–2–M:

1,2-Ethanediyl bismethanethiosulfonate

M–4–M:

1,4-Butanediyl bismethanethiosulfonate

M–6–M:

1,6-Hexanediyl bismethanethiosulfonate

M–8–M:

3,6-Dioxaoctane-1,8-diyl bismethanethiosulfonate

M–11–M:

3,6,9-Trioxaundecane-1,11-diyl bismethanethiosulfonate

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Acknowledgments

We are extremely thankful to William Zagotta who very generously supplied us with the clone of the CNGA1 and CNGA1cys-free channels and Claudio Anselmi for helpful discussions. This work was supported by a HFSP grant, a COFIN grant from the Italian Ministry, a grant from CIPE (GRAND FVG) and a FIRB grant from MIUR.

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  1. International School for Advanced Studies, via Beirut 2-4, 34014, Trieste, Italy

    Monica Mazzolini, Anil V. Nair & Vincent Torre

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  1. Monica Mazzolini
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  2. Anil V. Nair
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  3. Vincent Torre
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Correspondence to Vincent Torre.

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Mazzolini, M., Nair, A.V. & Torre, V. A comparison of electrophysiological properties of the CNGA1, CNGA1tandem and CNGA1cys-free Channels. Eur Biophys J 37, 947–959 (2008). https://doi.org/10.1007/s00249-008-0312-1

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  • DOI: https://doi.org/10.1007/s00249-008-0312-1

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