A ring of threonines in the inner vestibule of the pore of CNGA1 channels constitutes a binding site for permeating ions

• Published in: The Journal of physiology 2012-10, Vol.590 (20), p.5075-5090
• Main Authors: Marchesi, Arin, Mazzolini, Monica, Torre, Vincent
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Cattle; Cyclic Nucleotide-Gated Cation Channels - chemistry; Cyclic Nucleotide-Gated Cation Channels - physiology; In Vitro Techniques; Ions; Metals, Alkali - metabolism; Molecular and Cellular; Molecular Sequence Data; Mutagenesis, Site-Directed; Oocytes; Potassium Channels - chemistry; Sequence Alignment; Threonine - chemistry; Xenopus laevis
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.2012.238352

Key points •  Cyclic nucleotide‐gated (CNG) channels are multi‐ion channels showing the anomalous mole fraction effect (AMFE) in the presence of Li+ and Cs+ mixtures. •  We show that Cs+ ions at the intracellular side of the membrane block the entry of Na+ ions in a voltage dependent way. •  The blockage is relieved when Thr359 and Thr360 at the intracellular entrance of the selectivity filter are replaced with an alanine. Moreover, the AMFE in the presence of intracellular mixtures of Li+ and Cs+ is abolished in T360A mutant channels. •  We have identified a second binding site – composed by the ring of Thr360 at the intracellular vestibule – in the selectivity filter of CNG channels controlling monovalent cations selectivity and permeation. •  These results help us understand fundamental similarities and differences between the pore of CNG channels and K+ channels.   Cyclic nucleotide‐gated (CNG) channels and K+ channels have a significant sequence identity and are thought to share a similar 3D structure. K+ channels can accommodate simultaneously two or three permeating ions inside their pore and therefore are referred to as multi‐ion channels. Also CNGA1 channels are multi‐ion channels, as they exhibit an anomalous mole fraction effect (AMFE) in the presence of mixtures of 110 mm Li+ and Cs+ on the cytoplasmic side of the membrane. Several observations have identified the ring of Glu363 in the outer vestibule of the pore as one of the binding sites within the pore of CNGA1 channels. In the present work we identify a second binding site in the selectivity filter of CNGA1 channels controlling AMFE. Here, we show also that Cs+ ions at the intracellular side of the membrane block the entry of Na+ ions. This blockage is almost completely removed at high hyperpolarized voltages as expected if the Cs+ blocking site is located within the transmembrane electric field. Indeed, mutagenesis experiments show that the block is relieved when Thr359 and Thr360 at the intracellular entrance of the selectivity filter are replaced with an alanine. In T359A mutant channels AMFE in the presence of intracellular mixtures of Li+ and Cs+ is still present but is abolished in T360A mutant channels. These results suggest that the ring of Thr360 at the intracellular entrance of the selectivity filter forms another ion binding site in the CNGA1 channel. The two binding sites composed of the rings of Glu363 and Thr360 are not independent; in fact they mediate a powerful coupling be