Voltage Profile along the Permeation Pathway of an Open Channel
ABSTRACT For ion channels, the transmembrane potential plays a critical role by acting as a driving force for permeant ions. At the microscopic level, the transmembrane potential is thought to decay nonlinearly across the ion permeation pathway because of the irregular three-dimensional shape of the channel's pore. By taking advantage of the current structural and functional understanding of cyclic nucleotide-gated channels, in this study we experimentally explore the transmembrane potential's distribution across the open pore. As a readout for the voltage drop, we engineered cysteine residues along the selectivity filter and scanned the sensitivity of their modification rates by Ag(+) to the transmembrane potential. The experimental data, which indicate that the majority of the electric field drops across the selectivity filter, are in good agreement with continuum electrostatic calculations using a homology model of an open CNG channel. By focusing the transmembrane potential across the selectivity filter, the electromotive driving force is coupled with the movement of permeant ions in the filter, maximizing the efficiency of this process.
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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.The Journal of Physiology 12/2014; 593(4). DOI:10.1113/jphysiol.2014.284216 · 4.54 Impact Factor
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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.The Journal of Physiology 08/2012; 590(Pt 20):5075-90. DOI:10.1113/jphysiol.2012.238352 · 4.54 Impact Factor
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ABSTRACT: Understanding how ion channels open and close their pores is crucial for comprehending their physiological roles. We used intracellular quaternary ammonium blockers, electrophysiology and X-ray crystallography to locate the voltage-dependent gate in MthK potassium channels from Methanobacterium thermoautotrophicum. Blockers bind in an aqueous cavity between two putative gates: an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location-an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. Kinetic analysis of tetrabutylammonium block of single MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutyl antimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggest that MthK inactivation leads to conformational changes within the cavity and intracellular pore entrance.Nature Structural & Molecular Biology 12/2012; DOI:10.1038/nsmb.2473 · 11.63 Impact Factor