Insights into the Ion Selectivity Mechanism of CNG Channels from Mutants of NaK: Structural and Functional Studies

Abstract

Cyclic nucleotide-gated (CNG) channels are non-selective cation channels that play crucial roles in visual and olfactory signal transduction. They are members of the tetrameric cation channel family that include voltage-gated K+, Na+ and Ca2+ channels. However, while other members exhibit high degree of ion selectivity, CNG channels are noted for their lack of specificity. CNG channels conduct all alkali metal ions and some alkaline earths, most notably Ca2+. How the CNG channel pore can conduct these various cations which have substantially different ionic radii and formal charges is not well understood. Here we report high-resolution crystal structures of mutants of the NaK channel that mimic the selectivity filter of CNG channels, along with supporting functional analyses. Within the NaK selectivity filter (63TVGDGNFS70) the DGNFS sequence was replaced with ETPP, ETPT and DTPS, each of which represent a CNG α-subunit sequence. The mutant structures exhibit selectivity filter architecture and ion binding profiles different from either NaK or K+ channel structures, having three ion binding sites in their selectivity filters. Two of the sites correspond to sites 3 and 4 in KcsA and NaK, while the third site corresponds to site 2 in KcsA, but is a vestibule in NaK. Similar to CNG channels these mutants exhibit calcium binding, which depends on the presence of the conserved acidic residues (E or D). Mutating the acidic residues on these mutants to neutral residues (E◊Q or D◊N) abolishes calcium binding. Functional analyses using Rb-86 flux assay revealed ion conduction behavior similar to CNG channels. These results provide strong evidence that these NaK mutants exhibit the same properties of CNG channels in ion conduction and selectivity and their structures provide insight into understanding ion selectivity in CNG channels.