Structural insight into Ca
            <sup>2+</sup>
            specificity in tetrameric cation channels

Alam, Amer; Shi, Ning; Jiang, Youxing

doi:10.1073/pnas.0707324104

Cited by 36 publications

(45 citation statements)

References 32 publications

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“…Mutation of Asp 918 to Ala nearly eliminated Ca 2ϩ permeability of the channel, whereas substitution with the size-conserving uncharged Asn residue caused a more moderate decrease in Ca 2ϩ permeability, and substitution with the charge-conserving larger Glu residue led to an increase in Ca 2ϩ permeability. These results are inconsistent with a direct coordination of Ca 2ϩ by Asp 918 within the constrained environment of the ion permeation pathway and instead indicate that Asp 918 stabilizes the permeating Ca 2ϩ ions at a distance by electrostatics, like the stabilization of Ca 2ϩ by acidic residues in the pore of the NaKbc channel (48,50).…”

Section: Discussioncontrasting

confidence: 52%

“…Recent studies suggest that the pore of Ca 2ϩ -and Na ϩ -selective ion channels is structurally similar to that of K ϩ channels (48,49). The reported crystal structure of NaKbc, a nonselective cation channel from bacteria that shares homology with cyclic nucleotide-gated ion channels, shows that the carboxyl groups of conserved acidic residues do not line the permeation pathway, as previously assumed, and are instead tangential to it, acting through electrostatic interactions to stabilize permeating divalent cations (48,50). …”

Section: Discussionmentioning

confidence: 70%

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The Nociceptor Ion Channel TRPA1 Is Potentiated and Inactivated by Permeating Calcium Ions

Wang

Chang

Waters

et al. 2008

Journal of Biological Chemistry

243

312

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The transient receptor potential A1 (TRPA1) channel is the molecular target for environmental irritants and pungent chemicals, such as cinnamaldehyde and mustard oil. Extracellular Ca 2؉ is a key regulator of TRPA1 activity, both potentiating and subsequently inactivating it. In this report, we provide evidence that the effect of extracellular Ca 2؉ on these processes is indirect and can be entirely attributed to entry through TRPA1 and subsequent elevation of intracellular calcium. Specifically, we found that in a pore mutant of TRPA1, D918A, in which Ca 2؉ permeability was greatly reduced, extracellular Ca 2؉ produced neither potentiation nor inactivation. Both processes were restored by reducing intracellular Ca 2؉ buffering, which allowed intracellular Ca 2؉ levels to become elevated upon entry through D918A channels. Application of Ca 2؉ to the cytosolic face of excised patches was sufficient to produce both potentiation and inactivation of TRPA1 channels. Moreover, in whole cell recordings, elevation of intracellular Ca 2؉ by UV uncaging of 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA-potentiated TRPA1 currents. In addition, our data show that potentiation and inactivation are independent processes. TRPA1 currents could be inactivated by Mg 2؉ , Ba 2؉ , and Ca 2؉ but potentiated only by Ba 2؉ and Ca 2؉ . Saturating activation by cinnamaldehyde or mustard oil occluded potentiation but did not interfere with inactivation. Last, neither process was affected by mutation of a putative intracellular Ca 2؉ -binding EF-hand motif. In conclusion, we have further clarified the mechanisms of potentiation and inactivation of TRPA1 using the D918A pore mutant, an important tool for investigating the contribution of Ca 2؉ influx through TRPA1 to nociceptive signaling. Members of the transient receptor potential (TRP)2 family of ion channels that are expressed by sensory neurons in dorsal root and trigeminal ganglia serve as sensors for temperature and noxious stimuli (1, 2). Of these, TRPA1 is one of the key sensors for pungent chemicals and environmental irritants and is essential for behavioral responses of mice to conditions that evoke inflammatory pain (3-7). Inflammatory mediators, such as bradykinin, bind to G protein-coupled receptors on nociceptors, initiating a second messenger signaling cascade that leads to Ca 2ϩ influx mediated in part by the opening of Ca 2ϩ -permeable TRPA1 channels (5,8,9). TRPA1 is also activated directly by a wide range of chemicals that cause painful sensations, including food additives, such as mustard oil (MO), cinnamaldehyde (Cin), onion, raw garlic, and thyme; environmental irritants, such as formaldehyde and acrolein (a component of automobile exhaust); and products of oxidative stress (4, 8, 10 -16). Many of these chemicals activate TRPA1 by binding covalently to reactive cysteine residues in the amino terminus of the protein (17, 18), producing a modification of the channel that can last for more than 1 h and which leads to persistent activation of TRPA1 currents (18,19). Ca 2ϩ plays at...

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Section: Discussioncontrasting

confidence: 52%

Section: Discussionmentioning

confidence: 70%

The Nociceptor Ion Channel TRPA1 Is Potentiated and Inactivated by Permeating Calcium Ions

Wang

Chang

Waters

et al. 2008

Journal of Biological Chemistry

243

312

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“…NaK and its mutants for functional assays contained an extra Phe92-to-Ala mutation to enhance the single channel conductance. The proteins were purified in the detergent decyl maltoside and reconstituted in lipid vesicles composed of 3∶1 ratio of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylglycerol (Avanti Polar Lipids) as described (30,31). The wild-type MthK was analyzed in lipid bilayers as described (32).…”

Section: Methodsmentioning

confidence: 99%

Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites

Derebe¹,

Sauer²,

Zeng

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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111

184

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Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K þ channel selectivity filters. Combined with single channel electrophysiology, we show that only the channel with four ion binding sites is K þ selective, whereas those with two or three are nonselective and permeate Na þ and K þ equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel's selectivity properties and the presence of four sites in K þ channels is essential for highly selective and efficient permeation of K þ ions.nonselective cation channel | potassium channel T he structure determination of several K þ selective and, more recently, bacterial nonselective cation channels has, over the past decade, vastly increased our knowledge of ion selectivity mechanisms (1-8). In K þ channels, the TVGYG signature sequence has emerged as the fundamental element imparting high K þ over Na þ selectivity (9, 10), forming four contiguous and chemically equivalent K þ binding sites composed of backbone carbonyl oxygen atoms from filter residues together with the hydroxyl oxygen atoms from the conserved threonine. Conversely, the TVGDG filter sequence of the NaK channel from Bacillus cereus, although similar in length and amino acid composition to those of K þ channels, forms a nonselective filter preserving the two most intracellular sites of K þ channel filters along with a wide, water-filled vestibular region on the immediately extracellular side (7) (Fig. 1A). Debate lingers in the field about the underlying factors contributing to these selectivity differences. Although available structural studies of K þ channels seem to favor the classical snug-fit model to account for K þ over Na þ selectivity (11, 12), computational studies on K þ channel selectivity, in some cases using an isolated K þ binding site in their calculations, usually invoke one or more of the following concepts: the coordination number of the ion, chemistry of carbonyl oxygen ligands, intrinsic dynamism of the selectivity filter, solvent exposure of the ion binding sites, or the free energy landscapes of ion entrance and translocation in a multiion configuration (13-21). To further understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores whose selectivity filters contain three (equivalent to sites 2-4 or sites 1-3 of a K þ channel) or four (equivalent to sites 1-4 of a K þ channel) ion binding sites and determined their s...

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“…Similarly, these mutant structures were determined in an open conformation between resolutions of 1.58 and 1.95 Å (Table S1 and SI Materials and Methods). All chimeras used for functional studies contain an extra Phe to Ala mutation at a position equivalent to Phe92 of NaK in order to enhance the single channel conductance, and the proteins were reconstituted in lipid vesicles composed of 3∶1 ratio of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (Avanti Polar Lipids) at a protein/lipid ratio of 0.1 μg∕mg as described (21,32). Giant liposomes were obtained by air drying followed by rehydration.…”

Section: Methodsmentioning

confidence: 99%

“…A Glu-to-Asp mutation enhances Ca 2þ binding whereas a Glu-to-Asn mutation diminishes it. In the absence of CNG channel structures, structural insight into the molecular details underlying ion nonselectivity has been limited to K þ channel models (19,20) and, more recently, the prokaryotic nonselective cation channel NaK from Bacillus cereus (21,22). Although previous studies on NaK have yielded important insights into Ca 2þ binding in cation channels, they fall short of explaining several key mechanistic differences between NaK and CNG channels.…”

mentioning

confidence: 99%

Structural studies of ion permeation and Ca ²⁺ blockage of a bacterial channel mimicking the cyclic nucleotide-gated channel pore

Derebe¹,

Zeng

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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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 2þ 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 þ and K þ equally well, and exhibit the same Ca 2þ blockage and permeation properties. Their high-resolution structures reveal a hitherto unseen selectivity filter architecture comprising three contiguous ion binding sites in which Na þ and K þ bind with different ion-ligand geometries. Our structural analysis reveals that the conserved acidic residue in the filter is essential for Ca 2þ 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 þ channel models.

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Structural insight into Ca ²⁺ specificity in tetrameric cation channels

Cited by 36 publications

References 32 publications

The Nociceptor Ion Channel TRPA1 Is Potentiated and Inactivated by Permeating Calcium Ions

The Nociceptor Ion Channel TRPA1 Is Potentiated and Inactivated by Permeating Calcium Ions

Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites

Structural studies of ion permeation and Ca ²⁺ blockage of a bacterial channel mimicking the cyclic nucleotide-gated channel pore

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Structural insight into Ca 2+ specificity in tetrameric cation channels

Cited by 36 publications

References 32 publications

The Nociceptor Ion Channel TRPA1 Is Potentiated and Inactivated by Permeating Calcium Ions

The Nociceptor Ion Channel TRPA1 Is Potentiated and Inactivated by Permeating Calcium Ions

Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites

Structural studies of ion permeation and Ca 2+ blockage of a bacterial channel mimicking the cyclic nucleotide-gated channel pore

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Structural insight into Ca ²⁺ specificity in tetrameric cation channels

Structural studies of ion permeation and Ca ²⁺ blockage of a bacterial channel mimicking the cyclic nucleotide-gated channel pore