Single Molecule Force Spectroscopy of CNGA1 Channels In Situ

Abstract

Cyclic-nucleotide gated (CNG) channels are tetramers composed of distinct subunits. When the subunit CNGA1 is heterologously expressed in Xenopus laevis oocytes, functional channels are activated by cyclic nucleotides and have similar properties as that of native CNG channels. These channels belong to the family of voltage-gated ion channels and share a significant amino acid sequence identity with K+ channels suggesting a common ancestral three-dimensional structure. However, unlike K+ channels, the crystal structure of CNG channels is not known. In order to gain structural and functional information of these channels, we used single-molecule force spectroscopy (SMFS) to unfold individual CNGA1 subunit from Xenopus laevis oocyte's plasma membrane (“in situ”). We have also expressed CNGA1 subunit linked to a single molecule marker acting as a fingerprint in SMFS experiments, allowing the unequivocal recognition of spectra obtained from the unfolding of a single subunit of CNGA1 channels. Force traces of CNGA1 subunits showed a mechanical stability around 50pN. The comparison of contour length histograms in the closed and open states showed that in the open state, the cyclic-nucleotide binding (CNB) domain and the C-linker have a more defined folded structure and that significant conformational changes occur in the pore region upon gating.