Structural studies of ion permeation and Ca2+ blockage of a bacterial channel mimicking the cyclic nucleotide-gated channel pore.

Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9040, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 01/2011; 108(2):592-7. DOI: 10.1073/pnas.1013643108
Source: PubMed

ABSTRACT Cyclic nucleotide-gated (CNG) channels play an essential role in the visual and olfactory sensory systems and are ubiquitous in eukaryotes. Details of their underlying ion selectivity properties are still not fully understood and are a matter of debate in the absence of high-resolution structures. To reveal the structural mechanism of ion selectivity in CNG channels, particularly their Ca(2+) blockage property, we engineered a set of mimics of CNG channel pores for both structural and functional analysis. The mimics faithfully represent the CNG channels they are modeled after, permeate Na(+) and K(+) equally well, and exhibit the same Ca(2+) blockage and permeation properties. Their high-resolution structures reveal a hitherto unseen selectivity filter architecture comprising three contiguous ion binding sites in which Na(+) and K(+) bind with different ion-ligand geometries. Our structural analysis reveals that the conserved acidic residue in the filter is essential for Ca(2+) binding but not through direct ion chelation as in the currently accepted view. Furthermore, structural insight from our CNG mimics allows us to pinpoint equivalent interactions in CNG channels through structure-based mutagenesis that have previously not been predicted using NaK or K(+) channel models.

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    ABSTRACT: Ion channels control ionic fluxes across biological membranes by residing in any of three functionally distinct states: deactivated (closed), activated (open), or inactivated (closed). Unlike many of their cousin K(+) channels, cyclic nucleotide-gated (CNG) channels do not desensitize or inactivate. Using patch recording techniques, we show that when extracellular pH (pHo) is decreased from 7.4 to 6 or lower, wild-type CNGA1 channels inactivate in a voltage-dependent manner. pHo titration experiments show that at pHo < 7 the current-voltage relations are outwardly rectifying and that inactivation is coupled to current rectification. Single-channel recordings indicate that a fast mechanism of proton blockage underscore current rectification while inactivation arises from conformational changes downstream from protonation. Furthermore, mutagenesis and ionic substitution experiments highlight the role of the selectivity filter in current decline suggesting analogies with the C-type inactivation observed in K(+) channels. The analysis with Markovian models indicates that the non-independent binding of two protons within the transmembrane electrical field explains both the voltage-dependent blockage and the inactivation. Acidic pH by inhibiting the CNGA1 channels in a state-dependent manner may represent an unrecognized endogenous signal regulating CNG physiological functions in diverse tissues. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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    ABSTRACT: 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.
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