Mechanosensitive behavior of bacterial cyclic nucleotide gated (bCNG) ion channels: Insights into the mechanism of channel gating in the mechanosensitive channel of small conductance superfamily

We have recently identified and characterized the bacterial cyclic nucleotide gated (bCNG) subfamily of the larger mechanosensitive channel of small conductance (MscS) superfamily of ion channels. The channel domain of bCNG channels exhibits significant sequence homology to the mechanosensitive subfamily of MscS in the regions that have previously been used as a hallmark for channels that gate in response to mechanical stress. However, we have previously demonstrated that three of these channels are unable to rescue Escherichiacoli from osmotic downshock. Here, we examine an additional nine bCNG homologues and further demonstrate that the full-length bCNG channels are unable to rescue E. coli from hypoosmotic stress. However, limited mechanosensation is restored upon removal of the cyclic nucleotide binding domain. This indicates that the C-terminal domain of the MscS superfamily can drive channel gating and further highlight the ability of a superfamily of ion channels to be gated by multiple stimuli.     

Ss-bCNGa: a unique member of the bacterial cyclic nucleotide gated (bCNG) channel family that gates in response to mechanical tension

Bacterial cyclic nucleotide gated (bCNG) channels are generally a nonmechanosensitive subset of the mechanosensitive channel of small conductance (MscS) superfamily. bCNG channels are composed of an MscS channel domain, a linking domain, and a cyclic nucleotide binding domain. Among bCNG channels, the channel domain of Ss-bCNGa, a bCNG channel from Synechocystis sp. PCC 6803, is most identical to Escherichia coli (Ec) MscS. This channel also exhibits limited mechanosensation in response to osmotic downshock assays, making it the only known full-length bCNG channel to respond to hypoosmotic stress. Here, we compare and contrast the ability of Ss-bCNGa to gate in response to mechanical tension with Se-bCNG, a nonmechanosensitive bCNG channel, and Ec-MscS, a prototypical mechanosensitive channel. Compared with Ec-MscS, Ss-bCNGa only exhibits limited mechanosensation, which is most likely a result of the inability of Ss-bCNGa to form the strong lipid contacts needed for significant function. Unlike Ec-MscS, Ss-bCNGa displays a mechanical response that increases with protein expression level, which may result from channel clustering driven by interchannel cation?C?? interactions.     

环核苷酸门控离子通道门控的分子机理

环核苷酸门控离子通道(CNG)最广泛地分布于神经细胞。近年来关于 CNG 通道门控的分子机制的研究取得了很大的进步。研究表明, CNG 通道的组成及组装影响通道的特性及门控。近年来有关 CNG 突变体的研究及半胱氨酸残基亲和性的分析表明, 环核苷酸首先结合到 CNG 通道 C 端的环核苷酸结合域(CNBD)上引起 CNBD 空间构像改变, 然后 4 个亚单元发生空间构像的协调改变, CNG 通道开放。本文详细讨论了 CNG 通道的门控机制、各亚单元之间的相互作用、组装的过程及其空间构想的变化, 为 CNG 通道的进一步研究, 尤其是离子通道疾病方面提供理论指导。     

Recent characterizations of MscS and its homologs provide insight into the basis of ion selectivity in mechanosensitive channels

The bacterial mechanosensitive channel MscS provides an excellent model system for the study of mechanosensitivity and for investigations into the cellular response to hypoosmotic shock. Numerous studies have elucidated the structure, function and gating mechanism of Escherichia coli MscS, providing a wealth of information for the comparative analysis of MscS family members in bacteria, archaea, fungi and plants. We recently reported the electrophysiological characterization of MscS-Like (MSL)10, a MscS homolog from the model flowering plant Arabidopsis thaliana. Here we summarize our results and briefly compare MSL10 to previously described members of the MscS family. Finally, we comment on how this and other recently published studies illuminate the possible mechanisms by which ion selectivity is accomplished in this fascinating family of channels.     

Modification of cyclic nucleotide-gated ion channels by ultraviolet light

We irradiated cyclic nucleotide-gated ion channels in situ with ultraviolet light to probe the role of aromatic residues in ion channel function. UV light reduced the current through excised membrane patches from Xenopus oocytes expressing the alpha subunit of bovine retinal cyclic nucleotide-gated channels irreversibly, a result consistent with permanent covalent modification of channel amino acids by UV light. The magnitude of the current reduction depended only on the total photon dose delivered to the patches, and not on the intensity of the exciting light, indicating that the functionally important photochemical modification(s) occurred from an excited state reached by a one-photon absorption process. The wavelength dependence of the channels' UV light sensitivity (the action spectrum) was quantitatively consistent with the absorption spectrum of tryptophan, with a small component at long wavelengths, possibly due to cystine absorption. This spectral analysis suggests that UV light reduced the currents at most wavelengths studied by modifying one or more "target" tryptophans in the channels. Comparison of the channels' action spectrum to the absorption spectrum of tryptophan in various solvents suggests that the UV light targets are in a water-like chemical environment. Experiments on mutant channels indicated that the UV light sensitivity of wild-type channels was not conferred exclusively by any one of the 10 tryptophan residues in a subunit. The similarity in the dose dependences of channel current reduction and tryptophan photolysis in solution suggests that photochemical modification of a small number of tryptophan targets in the channels is sufficient to decrease the currents.     

Pore Structure of the Cys-loop Ligand-gated Ion Channels

The Cys-loop receptor family of ligand-gated ion channels (LGICs) play a key role in synaptic transmission in the central nervous system of animals. Recent advances have led to the elucidation of two crystal structures of related prokaryotic LGICs and the electron micrograph derived structure of the acetylcholine receptor from Torpedo marmorata. Here, we review the structural and biochemical data that form our understanding of the structure of the channel pore. We introduce original data from the glycine receptor using the substituted-cysteine accessibility technique and show that while the helical structure of the segment that surrounds the channel pore is generally agreed, the location of the channel gate, the pore diameter and the structure that forms the entry to the channel pore are likely to differ between receptors. The fundamental structural differences between anion and cation selective receptors and how these differences are related to the pore structure are also considered.     

Bacterial ion channels and their eukaryotic homologues.

Due to the relative ease of obtaining their crystal structures, bacterial ion channels provide a unique opportunity to analyse structure and function of their eukaryotic homologues. This review describes prokaryotic channels whose structures have been determined. These channels are KcsA, a bacterial homologue of eukaryotic potassium channels, MscL, a bacterial mechanosensitive ion channel and ClC0, a prokaryotic homologue of the eukaryotic ClC family of anion-selective channels. General features of their structure and function are described with a special emphasis on the advantages that these channels offer for understanding the properties of their eukaryotic homologues. We present amino-acid sequences of eukaryotic proteins related in their primary sequences to bacterial mechanosensitive channels. The usefulness of bacterial mechanosensitive channels for the studies on general principles of mechanosensation is discussed.     

Construction of a cyclic nucleotide-gated KcsA K+ channel

The ability of an ion channel to open in response to a defined stimulus is central to its function. In ligand-gated channels, pore opening is conferred through transduction of a conformational change in a gating domain to the helices of the pore. Here, we present the construction of a designed cyclic nucleotide-gated (CNG) channel, named KcsA-CNG, by addition of a prokaryotic cyclic nucleotide-binding domain to a KcsA-derived K+ channel. This channel is functional in lipid bilayers at physiological pH and has the combined properties of both of its parent-derived components. It conducts K+ and is blocked by the K+ channel inhibitors Na+ and agitoxin-2. Channel open times are increased by about two orders of magnitude compared to wild-type KcsA. The average number of open channels increases by approximately 50% upon addition of cAMP. Although the absolute open probabilities are somewhat variable from one channel to the next, the property of cyclic nucleotide sensitivity is very reproducible. An apparent Kd value of approximately 90 nM was estimated. The successful construction of a cyclic nucleotide-gated KcsA K+ channel suggests that it should be possible to produce channels that will respond to novel ligands.     

The influence of static magnetic fields on mechanosensitive ion channel activity in artificial liposomes

The influence of static magnetic fields (SMFs) on the activity of recombinant mechanosensitive ion channels (the bacterial mechanosensitive ion channel of large conductance—MscL) following reconstitution into artificial liposomes has been investigated. Preliminary findings suggest that exposure to 80-mT SMFs does not induce spontaneous MscL activation in the absence of mechanical stimulation. However, SMFs do appear to influence the open probability and single channel kinetics of MscL exposed to negative pipette pressure. Typical responses include an overall reduction in channel activity or an increased likelihood of channels becoming trapped open in sub-conducting states following exposure to SMFs. There is a delay in the onset of this effect and it is maintained throughout exposure. Generally, channel activity showed slow or limited recovery following removal of the magnetic field and responses to the magnetic were often reduced or abolished upon subsequent exposures. Pre-exposure of the liposomes to SMFs resulted in reduced sensitivity of MscL to negative pipette pressure, with higher pressures required to activate the channels. Although the mechanisms of this effect are not clear, our initial observations appear to support previous work showing that the effects of SMFs on ion channels may be mediated by changes in membrane properties due to anisotropic diamagnetism of lipid molecules.     

Primary structure of cAMP-gated channel from bovine olfactory epithelium

The complete amino-acid sequence of the bovine olfactory epithelium adenosine 3',5'cyclic monophosphate (cAMP)-gated channel has been determined by cloning and sequencing its cDNA. It exhibits a high degree of sequence homology with the cGMP-gated channel of rod photoreceptors, suggesting that cyclic nucleotide-gated channels fall into a new family of genetically related proteins.     

Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons.

We have cloned a functional cDNA encoding the cyclic nucleotide-gated channel selectively expressed in catfish olfactory sensory neurons. The cyclic nucleotide-gated channels share sequence and structural features with the family of voltage-gated ion channels. This homology is most evident in transmembrane region S4, the putative voltage sensor domain, and the H5 domain, thought to form the channel pore. We have characterized the single-channel properties of the cloned catfish channel and compared these properties with the channel in native catfish olfactory sensory neurons. The channel is activated equally well by cAMP and cGMP, shows only a slight voltage dependence of gating, and exhibits a pH- and voltage-dependent subconductance state similar to that observed for the voltage-gated L-type calcium channel.     

Sequence of events underlying the allosteric transition of rod cyclic nucleotide-gated channels

Activation of cyclic nucleotide-gated (CNG) ion channels involves a conformational change in the channel protein referred to as the allosteric transition. The amino terminal region and the carboxyl terminal cyclic nucleotide-binding domain of CNG channels have been shown to be involved in the allosteric transition, but the sequence of molecular events occurring during the allosteric transition is unknown. We recorded single-channel currents from bovine rod CNG channels in which mutations had been introduced in the binding domain at position 604 and/or the rat olfactory CNG channel amino terminal region had been substituted for the bovine rod amino terminal region. Using a hidden Markov modeling approach, we analyzed the kinetics of these channels activated by saturating concentrations of cGMP, cIMP, and cAMP. We used thermodynamic mutant cycles to reveal an interaction during the allosteric transition between the purine ring of the cyclic nucleotides and the amino acid at position 604 in the binding site. We found that mutations at position 604 in the binding domain alter both the opening and closing rate constants for the allosteric transition, indicating that the interactions between the cyclic nucleotide and this amino acid are partially formed at the time of the transition state. In contrast, the amino terminal region affects primarily the closing rate constant for the allosteric transition, suggesting that the state-dependent stabilizing interactions between amino and carboxyl terminal regions are not formed at the time of the transition state for the allosteric transition. We propose that the sequence of events that occurs during the allosteric transition involves the formation of stabilizing interactions between the purine ring of the cyclic nucleotide and the amino acid at position 604 in the binding domain followed by the formation of stabilizing interdomain interactions.     

Influence of permeant ions on gating in cyclic nucleotide-gated channels

Cyclic nucleotide-gated channels are key components in the transduction of visual and olfactory signals where their role is to respond to changes in the intracellular concentration of cyclic nucleotides. Although these channels poorly select between physiologically relevant monovalent cations, the gating by cyclic nucleotide is different in the presence of Na(+) or K(+) ions. This property was investigated using rod cyclic nucleotide-gated channels formed by expressing the subunit 1 (or alpha) in HEK293 cells. In the presence of K(+) as the permeant ion, the affinity for cGMP is higher than the affinity measured in the presence of Na(+). At the single channel level, subsaturating concentrations of cGMP show that the main effect of the permeant K(+) ions is to prolong the time channels remain open without major changes in the shut time distribution. In addition, the maximal open probability was higher when K(+) was the permeant ion (0.99 for K(+) vs. 0.95 for Na(+)) due to an increase in the apparent mean open time. Similarly, in the presence of saturating concentrations of cAMP, known to bind but unable to efficiently open the channel, permeant K(+) ions also prolong the time channels visit the open state. Together, these results suggest that permeant ions alter the stability of the open conformation by influencing of the O-->C transition.     

Plants do it differently. A new basis for potassium/sodium selectivity in the pore of an ion channel

Understanding of the molecular architecture necessary for selective K(+) permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K(+) channel KcsA, and structure:function studies of cloned animal K(+) channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na(+) and K(+) permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K(+)-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na(+) and K(+) permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na(+). Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K(+) and Na(+) permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K(+) over Na(+) conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K(+) channels that has been identified as the critical basis for K(+) over Na(+) permeation through the pore of ion channels.     

Electrophysiological analysis of cloned cyclic nucleotide-gated ion channels

Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K(+) and other monovalent cations, but exclude Na(+); this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K(+)-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotide-gated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K(+) currents.     

Mass spectrometry-based phosphoproteomics reveals multisite phosphorylation on mammalian brain voltage-gated sodium and potassium channels

Voltage-gated sodium and potassium channels underlie electrical activity of neurons, and are dynamically regulated by diverse cell signaling pathways that ultimately exert their effects by altering the phosphorylation state of channel subunits. Recent mass spectrometric-based studies have led to a new appreciation of the extent and nature of phosphorylation of these ion channels in mammalian brain. This has allowed for new insights into how neurons dynamically regulate the localization, activity and expression through multisite ion channel phosphorylation.     

Extracellular proton sensing of the rat gustatory cyclic nucleotide-gated channel

Elevations of the intracellular levels of cyclic nucleotides appear to cause the cation influx through gustatory cyclic nucleotide-gated (CNGgust) channels expressed in taste cells. Although changes in the oral pH may directly regulate the activity of the CNGgust channel, the mechanism of pH-dependent control of the channel is not understood. In the present study, we combined the whole-cell patch-clamp recording and the site-directed mutagenesis to investigate the effect of extracellular pH on the ion permeation through CNGgust channels expressed in HEK293 cells. Extracellular acidification strongly inhibited ion permeation through open CNGgust channels. Mutation of Glu(289) remarkably attenuated the pH-dependence of the channel, suggesting that Glu(289) in the pore-forming region is a major proton acceptor site. However, the mutant E289A-CNGgust channel possesses the other residual protonation/deprotonation site. The channel activity, tightly regulated by pH(o) and [cNMP](i), suggests the involvement of its pH(o)-dependent ion permeation in taste signal transduction events.     

Identification of a functionally essential amino acid for Arabidopsis cyclic nucleotide gated ion channels using the chimeric AtCNGC11/12 gene

We used the chimeric Arabidopsis cyclic nucleotide-gated ion channel AtCNGC11/12 to conduct a structure-function study of plant cyclic nucleotide-gated ion channels (CNGCs). AtCNGC11/12 induces multiple pathogen resistance responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22). A genetic screen for mutants that suppress cpr22-conferred phenotypes identified an intragenic mutant, #73, which has a glutamate to lysine substitution (E519K) at the beginning of the eighth beta-sheet of the cyclic nucleotide-binding domain in AtCNGC11/12. The #73 mutant is morphologically identical to wild-type plants and has lost cpr22-related phenotypes including spontaneous cell death and enhanced pathogen resistance. Heterologous expression analysis using a K(+)-uptake-deficient yeast mutant revealed that this Glu519 is important for AtCNGC11/12 channel function, proving that the occurrence of cpr22 phenotypes requires active channel function of AtCNGC11/12. Additionally, Glu519 was also found to be important for the function of the wild-type channel AtCNGC12. Computational structural modeling and in vitro cAMP-binding assays suggest that Glu519 is a key residue for the structural stability of AtCNGCs and contributes to the interaction of the cyclic nucleotide-binding domain and the C-linker domain, rather than the binding of cAMP. Furthermore, a mutation in the alpha-subunit of the human cone receptor CNGA3 that causes total color blindness aligned well to the position of Glu519 in AtCNGC11/12. This suggests that AtCNGC11/12 suppressors could be a useful tool for discovering important residues not only for plant CNGCs but also for CNGCs in general.     

Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel

Cyclic nucleotide-gated (cng) non-selective cation channels have been cloned from a number of animal systems. These channels are characterized by direct gating upon cAMP or cGMP binding to the intracellular portion of the channel protein, which leads to an increase in channel conductance. Animal cng channels are involved in signal transduction systems; they translate stimulus-induced changes in cytosolic cyclic nucleotide into altered cell membrane potential and/or cation flux as part of a signal cascade pathway. Putative plant homologs of animal cng channels have been identified. However, functional characterization (i.e. demonstration of cyclic-nucleotide-dependent ion currents) of a plant cng channel has not yet been accomplished. We report the cloning and first functional characterization of a plant member of this family of ion channels. The Arabidopsis cDNA AtCNGC2 encodes a polypeptide with deduced homology to the alpha-subunit of animal channels, and facilitates cyclic nucleotide-dependent cation currents upon expression in a number of heterologous systems. AtCNGC2 expression in a yeast mutant lacking a low-affinity K(+) uptake system complements growth inhibition only when lipophilic cyclic nucleotides are present in the culture medium. Voltage clamp analysis indicates that Xenopus laevis oocytes injected with AtCNGC2 cRNA demonstrate cyclic-nucleotide-dependent, inward-rectifying K(+) currents. Human embryonic kidney cells (HEK293) transfected with AtCNGC2 cDNA demonstrate increased permeability to Ca(2+) only in the presence of lipophilic cyclic nucleotides. The evidence presented here supports the functional classification of AtCNGC2 as a cyclic-nucleotide-gated cation channel, and presents the first direct evidence (to our knowledge) identifying a plant member of this ion channel family.     

Cystic fibrosis transmembrane conductance regulator. Physical basis for lyotropic anion selectivity patterns

The selectivity of Ca2+ over Na+ is approximately 3.3-fold larger in cGMP-gated channels of cone photoreceptors than in those of rods when measured under saturating cGMP concentrations, where the probability of channel opening is 85-90%. Under physiological conditions, however, the probability of opening of the cGMP-gated channels ranges from its largest value in darkness of 1-5% to essentially zero under continuous, bright illumination. We investigated the ion selectivity of cGMP-gated channels as a function of cyclic nucleotide concentration in membrane patches detached from the outer segments of rod and cone photoreceptors and have found that ion selectivity is linked to gating. We determined ion selectivity relative to Na+ (PX/PNa) from the value of reversal potentials measured under ion concentration gradients. The selectivity for Ca2+ over Na+ increases continuously as the probability of channel opening rises. The dependence of PCa/PNa on cGMP concentration, in both rods and cones, is well described by the same Hill function that describes the cGMP dependence of current amplitude. At the cytoplasmic cGMP concentrations expected in dark-adapted intact photoreceptors, PCa/PNa in cone channels is approximately 7.4-fold greater than that in rods. The linkage between selectivity and gating is specific for divalent cations. The selectivity of Ca2+ and Sr2+ changes with cGMP concentration, but the selectivity of inorganic monovalent cations, Cs+ and NH4+, and organic cations, methylammonium+ and dimethylammonium+, is invariant with cGMP. Cyclic nucleotide-gated channels in rod photoreceptors are heteromeric assemblies of alpha and beta subunits. The maximal PCa/PNa of channels formed from alpha subunits of bovine rod channels is less than that of heteromeric channels formed from alpha and beta subunits. In addition, Ca2+ is a more effective blocker of channels formed by alpha subunits than of channels formed by alpha and beta subunits. The cGMP-dependent shift in divalent cation selectivity is a property of alphabeta channels and not of channels formed from alpha subunits alone.