title: P-loop residues critical for selectivity in K channels fail to confer selectivity to rabbit HCN4 channels.
abstract: HCN channels are thought to be structurally similar to Kv channels, but show much lower selectivity for K+. The approximately 3.3 A selectivity filter of K+ channels is formed by the pore-lining sequence XT(V/I)GYG, with X usually T, and is held stable by key residues in the P-loop. Differences in the P-loop sequence of HCN channels (eg. the pore-lining sequence L478C479IGYG) suggest these residues could account for differences in selectivity between these channel families. Despite being expressed, L478T/C479T HCN4 channels did not produce current. Since threonine in the second position is highly conserved in K+ channels, we also studied C479T channels. Based on permeability ratios (PX/PK), C479T HCN4 channels (K+(1)>Rb+(0.85)>Cs+(0.59)>Li+(0.50)>or=Na+(0.49)) were less selective than WT rabbit HCN4 (K+(1)>Rb+(0.48)>Cs+(0.31)>or=Na+(0.29)>Li+(0.03)), indicating that the TIGYG sequence is insufficient to confer K+ selectivity to HCN channels. C479T HCN4 channels had an increased permeability to large organic cations than WT HCN4 channels, as well as increased unitary K+ conductance, and altered channel gating. Collectively, these results suggest that HCN4 channels have larger pores than K+ channels and replacement of the cysteine at position 479 with threonine further increases pore size. Furthermore, selected mutations in other regions linked previously to pore stability in K+ channels (ie. S475D, S475E and F471W/K472W) were also unable to confer K+ selectivity to C479T HCN4 channels. Our findings establish the presence of the TIGYG pore-lining sequence does not confer K+ selectivity to rabbit HCN4 channels, and suggests that differences in selectivity of HCN4 versus K+ channels originate from differences outside the P-loop region.
authors.affiliation: Department of Physiology, Heart and Stroke/Richard Lewar Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Nazzareno
authors.initials: N
authors.lastname: D'Avanzo
authors.forename: Roman
authors.initials: R
authors.lastname: Pekhletski
authors.forename: Peter H
authors.initials: PH
authors.lastname: Backx
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000502:physiology*; D016296:Mutagenesis; D009154:Mutation; D010539:Permeability; D011188:Potassium / Q000737:chemistry; D015221:Potassium Channels / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D017386:Sequence Homology, Amino Acid
chemical_list: D009124:Muscle Proteins; D015221:Potassium Channels; D011188:Potassium