Selectivity filter gating in large-conductance Ca(2+)-activated K+ channels

Membrane voltage controls the passage of ions through voltage-gated K (K(v)) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of K(v) channels is well established, it is not clear if such a cytoplasmic gate exists in all K(+) channels. Some studies on large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker "ball" peptide (BP) on BK channels with either K(+) or Rb(+) as the permeant ion. When tested in K(+) solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb(+) replaced K(+) as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these K(v) channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating.     

Stabilization of the Conductive Conformation of a Voltage-gated K+ (Kv) Channel: THE LID MECHANISM*

Voltage-gated K⁺ (Kv) channels are molecular switches that sense membrane potential and in response open to allow K⁺ ions to diffuse out of the cell. In these proteins, sensor and pore belong to two distinct structural modules. We previously showed that the pore module alone is a robust yet dynamic structural unit in lipid membranes and that it senses potential and gates open to conduct K⁺ with unchanged fidelity. The implication is that the voltage sensitivity of K⁺ channels is not solely encoded in the sensor. Given that the coupling between sensor and pore remains elusive, we asked whether it is then possible to convert a pore module characterized by brief openings into a conductor with a prolonged lifetime in the open state. The strategy involves selected probes targeted to the filter gate of the channel aiming to modulate the probability of the channel being open assayed by single channel recordings from the sensorless pore module reconstituted in lipid bilayers. Here we show that the premature closing of the pore is bypassed by association of the filter gate with two novel open conformation stabilizers: an antidepressant and a peptide toxin known to act selectively on Kv channels. Such stabilization of the conductive conformation of the channel is faithfully mimicked by the covalent attachment of fluorescein at a cysteine residue selectively introduced near the filter gate. This modulation prolongs the occupancy of permeant ions at the gate. It is this longer embrace between ion and gate that we conjecture underlies the observed stabilization of the conductive conformation. This study provides a new way of thinking about gating.     

Ion binding in the open HCN pacemaker channel pore: fast mechanisms to shape "slow" channels

I(H) pacemaker channels carry a mixed monovalent cation current that, under physiological ion gradients, reverses at approximately -34 mV, reflecting a 4:1 selectivity for K over Na. However, I(H) channels display anomalous behavior with respect to permeant ions such that (a) open channels do not exhibit the outward rectification anticipated assuming independence; (b) gating and selectivity are sensitive to the identity and concentrations of externally presented permeant ions; (c) the channels' ability to carry an inward Na current requires the presence of external K even though K is a minor charge carrier at negative voltages. Here we show that open HCN channels (the hyperpolarization-activated, cyclic nucleotide sensitive pore forming subunits of I(H)) undergo a fast, voltage-dependent block by intracellular Mg in a manner that suggests the ion binds close to, or within, the selectivity filter. Eliminating internal divalent ion block reveals that (a) the K dependence of conduction is mediated via K occupancy of site(s) within the pore and that asymmetrical occupancy and/or coupling of these sites to flux further shapes ion flow, and (b) the kinetics of equilibration between K-vacant and K-occupied states of the pore (10-20 micros or faster) is close to the ion transit time when the pore is occupied by K alone ( approximately 0.5-3 micros), a finding that indicates that either ion:ion repulsion involving Na is adequate to support flux (albeit at a rate below our detection threshold) and/or the pore undergoes rapid, permeant ion-sensitive equilibration between nonconducting and conducting configurations. Biophysically, further exploration of the Mg site and of interactions of Na and K within the pore will tell us much about the architecture and operation of this unusual pore. Physiologically, these results suggest ways in which "slow" pacemaker channels may contribute dynamically to the shaping of fast processes such as Na-K or Ca action potentials.     

A binding-block ion selective mechanism revealed by a Na/K selective channel

Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na⁺/K⁺ cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaI^Met158) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaI^Met158 facilitate Na⁺/K⁺ pass through, which was defined as bindingblock mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.     

Anomalous Effect of Permeant Ion Concentration on Peak Open Probability of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. Decreasing extracellular permeant ion concentration decreases outward Na⁺ current at positive voltages while increasing the driving force for the current. This anomalous effect of permeant ion concentration, especially obvious in a mutant (F1485Q) in which fast inactivation is partially abolished, is due to an alteration of open probability. The effect is only observed when a highly permeant cation (Na⁺, Li⁺, or hydrazinium) is substituted for a relatively impermeant cation (K⁺, Rb⁺, Cs⁺, N -methylglucamine, Tris, choline, or tetramethylammonium). With high concentrations of extracellular permeant cations, the peak open probability of Na⁺ channels increases with depolarization and then saturates at positive voltages. By contrast, with low concentrations of permeant ions, the open probability reaches a maximum at approximately 0 mV and then decreases with further depolarization. There is little effect of permeant ion concentration on activation kinetics at depolarized voltages. Furthermore, the lowered open probability caused by a brief depolarization to +60 mV recovers within 5 ms upon repolarization to −140 mV, indicative of a gating process with rapid kinetics. Tail currents at reduced temperatures reveal the rapid onset of this gating process during a large depolarization. A large depolarization may drive a permeant cation out of a site within the extracellular mouth of the pore, reducing the efficiency with which the channel opens.     

Simulation of the effect of an external GHz electric field on the potential energy profile of Ca2+ ions in the selectivity filter of the CaVAb channel

Ca_V channels are transmembrane proteins that mediate and regulate ion fluxes across cell membranes, and they are activated in response to action potentials to allow Ca²⁺ influx. Since ion channels are composed of charge or polar groups, an external alternating electric field may affect the ion‐selective membrane transport and the performance of the channel. In this article, we have investigated the effect of an external GHz electric field on the dynamics of calcium ions in the selectivity filter of the Ca_VAb channel. Molecular dynamics (MD) simulations and the potential of mean force (PMF) calculations were carried out, via the umbrella sampling method, to determine the free energy profile of Ca²⁺ ions in the Ca_VAb channels in presence and absence of an external field. Exposing Ca_VAb channel to 1, 2, 3, 4, and 5 GHz electric fields increases the depth of the potential energy well and this may result in an increase in the affinity and strength of Ca²⁺ ions to binding sites in the selectivity filter the channel. This increase of strength of Ca²⁺ ions binding in the selectivity filter may interrupt the mechanism of Ca²⁺ ion conduction, and leads to a reduction of Ca²⁺ ion permeation through the Ca_VAb channel.     

Ion selectivity mechanism in a bacterial pentameric ligand-gated ion channel

The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high-resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential-of-mean-force profiles for sodium and chloride ions inside the transmembrane region. Our calculations reveal that the GLIC channel is open for a sodium ion to transport, but presents a ∼11 kcal/mol free energy barrier for a chloride ion. Our collective findings identify three distinct contributions to the observed preference for the permeant ions. First, there is a substantial contribution due to a ring of negatively charged glutamate residues (E-2′) at the narrow intracellular end of the channel. The negative electrostatics of this region and the ability of the glutamate side chains to directly bind cations would strongly favor the passage of sodium ions while hindering translocation of chloride ions. Second, our results imply a significant hydrophobic contribution to selectivity linked to differences in the desolvation penalty for the sodium versus chloride ions in the central hydrophobic region of the pore. This hydrophobic contribution is evidenced by the large free energy barriers experienced by Cl⁻ in the middle of the pore for both GLIC and the E-2′A mutant. Finally, there is a distinct contribution arising from the overall negative electrostatics of the channel.     

Binding of kappa-conotoxin PVIIA to Shaker K+ channels reveals different K+ and Rb+ occupancies within the ion channel pore

The x-ray structure of the KcsA channel at different [K(+)] and [Rb(+)] provided insight into how K(+) channels might achieve high selectivity and high K(+) transit rates and showed marked differences between the occupancies of the two ions within the ion channel pore. In this study, the binding of kappa-conotoxin PVIIA (kappa-PVIIA) to Shaker K(+) channel in the presence of K(+) and Rb(+) was investigated. It is demonstrated that the complex results obtained were largely rationalized by differences in selectivity filter occupancy of this 6TM channels as predicted from the structural work on KcsA. kappa-PVIIA inhibition of the Shaker K(+) channel differs in the closed and open state. When K(+) is the only permeant ion, increasing extracellular [K(+)] decreases kappa-PVIIA affinity for closed channels by decreasing the "on" binding rate, but has no effect on the block of open channels, which is influenced only by the intracellular [K(+)]. In contrast, extracellular [Rb(+)] affects both closed- and open-channel binding. As extracellular [Rb(+)] increases, (a) binding to the closed channel is slightly destabilized and acquires faster kinetics, and (b) open channel block is also destabilized and the lowest block seems to occur when the pore is likely filled only by Rb(+). These results suggest that the nature of the permeant ions determines both the occupancy and the location of the pore site from which they interact with kappa-PVIIA binding. Thus, our results suggest that the permeant ion(s) within a channel pore can determine its functional and pharmacological properties.     

The pore structure and gating mechanism of K2P channels

Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability. However, the structure of these channels and their gating mechanism, in particular the role of the bundle-crossing gate, are not well understood. Here, we report that quaternary ammonium (QA) ions bind with high-affinity deep within the pore of TREK-1 and have free access to their binding site before channel activation by intracellular pH or pressure. This demonstrates that, unlike most other K(+) channels, the bundle-crossing gate in this K2P channel is constitutively open. Furthermore, we used QA ions to probe the pore structure of TREK-1 by systematic scanning mutagenesis and comparison of these results with different possible structural models. This revealed that the TREK-1 pore most closely resembles the open-state structure of KvAP. We also found that mutations close to the selectivity filter and the nature of the permeant ion profoundly influence TREK-1 channel gating. These results demonstrate that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter and do not involve gating at the cytoplasmic bundle crossing.     

KcsA 通道对Na+、K+及Rb+离子选择性的统计热力学研究

钾离子的通透率至少比钠离子的通透率大10000倍,这个问题至今没有很好地解决.为了在分子水平阐释钾离子通道的选择性机制,以KcsA钾通道X射线衍射结构为基础,采用密度泛函理论计算了不同离子在离子通道中的位能.计算结果表明,Rb+离子具有与K+离子相类似的位能曲线,但是其在通透过程遇到的位垒要比K+离子的位垒高,因而所对应的通透率也就小于钾离子的通透率,而钠离子的的通透率仅仅是钾离子通透率的0.0067%.文中所涉及的系统仅仅包含269个原子,而用分子动力学虽然也可以得到相近的结果,但是它的系统大小为41 000个原子.     

The occupancy of ions in the K+ selectivity filter: charge balance and coupling of ion binding to a protein conformational change underlie high conduction rates

Potassium ions diffuse across the cell membrane in a single file through the narrow selectivity filter of potassium channels. The crystal structure of the KcsA K+ channel revealed the chemical structure of the selectivity filter, which contains four binding sites for K+. In this study, we used Tl+ in place of K+ to address the question of how many ions bind within the filter at a given time, i.e. what is the absolute ion occupancy? By refining the Tl+ structure against data to 1.9A resolution with an anomalous signal, we determined the absolute occupancy of Tl+. Then, by comparing the electron density of Tl+ with that of K+, Rb+ and Cs+, we estimated the absolute occupancy of these three ions. We further analyzed how the ion occupancy affects the conformation of the selectivity filter by analyzing the structure of KcsA at different concentrations of Tl+. Our results indicate that the average occupancy for each site in the selectivity filter is about 0.63 for Tl+ and 0.53 for K+. For K+, Rb+ and Cs+, the total number of ions contained within four sites in the selectivity filter is about two. At low concentrations of permeant ion, the number of ions drops to one in association with a conformational change in the selectivity filter. We conclude that electrostatic balance and coupling of ion binding to a protein conformational change underlie high conduction rates in the setting of high selectivity.     

Tail end of the s6 segment: role in permeation in shaker potassium channels

The permeation pathway in voltage-gated potassium channels has narrow constrictions at both the extracellular and intracellular ends. These constrictions might limit the flux of cations from one side of the membrane to the other. The extracellular constriction is the selectivity filter, whereas the intracellular bundle crossing is proposed to act as the activation gate that opens in response to a depolarization. This four-helix bundle crossing is composed of S6 transmembrane segments, one contributed by each subunit. Here, we explore the cytoplasmic extension of the S6 transmembrane segment of Shaker potassium channels, just downstream from the bundle crossing. We substituted cysteine for each residue from N482 to T489 and determined the amplitudes of single channel currents and maximum open probability (P(o,max)) at depolarized voltages using nonstationary noise analysis. One mutant, F484C, significantly reduces P(o,max), whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (gamma). Mutations of residue Y485 have no effect on the Rb(+)/K(+) selectivity, suggesting a local effect on gamma rather than an allosteric effect on the selectivity filter. Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate. Replacing Rb(+) ions for K(+) ions reduces the amplitude of single channel currents and makes gamma insensitive to mutations of Y485. These results suggest that Rb(+) ions increase an extracellular energy barrier, presumably at the selectivity filter, thus making it rate limiting for flux of permeant ions. These results indicate that S6(T) residues have an influence on the conformation of the open activation gate, reflected in both the stability of the open state and the energy barriers it presents to ions.     

A flexible GAS belt responds to pore mutations changing the ion selectivity of proton-gated channels

Proton-gated ion channels conduct mainly Na⁺ to induce postsynaptic membrane depolarization. Finding the determinants of ion selectivity requires knowledge of the pore structure in the open conformation, but such information is not yet available. Here, the open conformation of the hASIC1a channel was computationally modeled, and functional effects of pore mutations were analyzed in light of the predicted structures. The open pore structure shows two constrictions of similar diameter formed by the backbone of the GAS belt and, right beneath it, by the side chains of H28 from the reentrant loop. Models of nonselective mutant channels, but not those that maintain ion selectivity, predict enlargement of the GAS belt, suggesting that this motif is quite flexible and that the loss of stabilizing interactions in the central pore leads to changes in size/shape of the belt. Our results are consistent with the “close-fit” mechanism governing selectivity of hASIC1a, wherein the backbone of the GAS substitutes at least part of the hydration shell of a permeant ion to enable crossing the pore constriction.     

Gating the glutamate gate of CLC-2 chloride channel by pore occupancy

CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage–sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H⁺. Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl⁻, Br⁻, SCN⁻, and I⁻) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pH_e = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl⁻]_i under unlikely protonation conditions (pH_e = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H⁺ plays a minor role in dislodging the glutamate gate.     

KcsA通道对Na+、K+及Rb+离子选择性的统计热力学研究

钾离了的通透率至少比钠离子的通透率大10000倍,这个问题至今没有很好地解决,为了存分子水平阐释钾离子通道的选择性机制,以KcsA钾通道X射线衍射结构为基础,采用密度泛函理论计算了不同离子在离子通道中的位能．计算结果表明,Rb^ 离子具有与K^ 离子相类似的位能曲线,但是其在通透过程遇到的位垒要比K^ 离子的位垒高,因而所对应的通透率也就小十钾离子的通透率,而钠离子的的通透率仅仅足钾离子通透率的0．0067％．文中所涉及的系统仪仅包含269个原子,而用分子动力学虽然也可以得到相近的结果,但是它的系统火小为41000个原子．     

Ligand-gated ion channels: mechanisms underlying ion selectivity

Anion/cation selectivity is a critical property of ion channels and underpins their physiological function. Recently, there have been numerous mutagenesis studies, which have mapped sites within the ion channel-forming segments of ligand-gated ion channels that are determinants of the ion selectivity. Site-directed mutations to specific amino acids within or flanking the M2 transmembrane segments of the anion-selective glycine, GABA(A) and GABA(C) receptors and the cation-selective nicotinic acetylcholine and serotonin (type 3) receptors have revealed discrete, equivalent regions within the ion channel that form the principal selectivity filter, leading to plausible molecular mechanisms and mathematical models to describe how ions preferentially permeate these channels. In particular, the dominant factor determining anion/cation selectivity seems to be the sign and exposure of charged amino acids lining the selectivity filter region of the open channel. In addition, the minimum pore diameter, which can be influenced by the presence of a local proline residue, also makes a contribution to such ion selectivity in LGICs with smaller diameters increasing anion/cation selectivity and larger ones decreasing it.     

Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells

Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.     

Ion interactions in the high-affinity binding locus of a voltage-gated Ca(2+) channel

The selectivity filter of voltage-gated Ca(2+) channels is in part composed of four Glu residues, termed the EEEE locus. Ion selectivity in Ca(2+) channels is based on interactions between permeant ions and the EEEE locus: in a mixture of ions, all of which can pass through the pore when present alone, those ions that bind weakly are impermeant, those that bind more strongly are permeant, and those that bind more strongly yet act as pore blockers as a consequence of their low rate of unbinding from the EEEE locus. Thus, competition among ion species is a determining feature of selectivity filter function in Ca(2+) channels. Previous work has shown that Asp and Ala substitutions in the EEEE locus reduce ion selectivity by weakening ion binding affinity. Here we describe for wild-type and EEEE locus mutants an analysis at the single channel level of competition between Cd(2+), which binds very tightly within the EEEE locus, and Ba(2+) or Li(+), which bind less tightly and hence exhibit high flux rates: Cd(2+) binds to the EEEE locus approximately 10(4)x more tightly than does Ba(2+), and approximately 10(8)x more tightly than does Li(+). For wild-type channels, Cd(2+) entry into the EEEE locus was 400x faster when Li(+) rather than Ba(2+) was the current carrier, reflecting the large difference between Ba(2+) and Li(+) in affinity for the EEEE locus. For the substitution mutants, analysis of Cd(2+) block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate. Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs. Ala), but was instead determined by the valence and affinity of the current-carrying ion (Ba(2+) vs. Li(+)). The dependence of Cd(2+) block kinetics upon properties of the current-carrying ion can be understood by considering the number of EEEE locus oxygen atoms available to interact with the different ion pairs.     

Comparative study of the energetics of ion permeation in Kv1.2 and KcsA potassium channels

Biological ion channels rely on a multi-ion transport mechanism for fast yet selective permeation of ions. The crystal structure of the KcsA potassium channel provided the first microscopic picture of this process. A similar mechanism is assumed to operate in all potassium channels, but the validity of this assumption has not been well investigated. Here, we examine the energetics of ion permeation in Shaker Kv1.2 and KcsA channels, which exemplify the six-transmembrane voltage-gated and two-transmembrane inward-rectifier channels. We study the feasibility of binding a third ion to the filter and the concerted motion of ions in the channel by constructing the potential of mean force for K⁺ ions in various configurations. For both channels, we find that a pair of K⁺ ions can move almost freely within the filter, but a relatively large free-energy barrier hinders the K⁺ ion from stepping outside the filter. We discuss the effect of the CMAP dihedral energy correction that was recently incorporated into the CHARMM force field on ion permeation dynamics.     

Extracellular protons titrate voltage gating of a ligand-gated ion channel

Cyclic nucleotide–gated channels mediate transduction of light into electric signals in vertebrate photoreceptors. These channels are primarily controlled by the binding of intracellular cyclic GMP (cGMP). Glutamate residue 363 near the extracellular end of the ion selectivity filter interacts with the pore helix and helps anchor the filter to the helix. Disruption of this interaction by mutations renders the channels essentially fully voltage gated in the presence of saturating concentrations of cGMP. Here, we find that lowering extracellular pH makes the channels conduct in an extremely outwardly rectifying manner, as does a neutral glutamine substitution at E363. A pair of cysteine mutations, E363C and L356C (the latter located midway the pore helix), largely eliminates current rectification at low pH. Therefore, this low pH-induced rectification primarily reflects voltage-dependent gating involving the ion selectivity filter rather than altered electrostatics around the external opening of the ion pore and thus ion conduction. It then follows that protonation of E363, like the E363Q mutation, disrupts the attachment of the selectivity filter to the pore helix. Loosening the selectivity filter from its surrounding structure shifts the gating equilibrium toward closed states. At low extracellular pH, significant channel opening occurs only when positive voltages drive the pore from a low probability open conformation to a second open conformation. Consequently, at low extracellular pH the channels become practically fully voltage gated, even in the presence of a saturating concentration of cGMP.