Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel

Root, Michael J.; MacKinnon, Roderick

doi:10.1016/0896-6273(93)90150-p

Cited by 214 publications

(176 citation statements)

References 21 publications

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“…The pattern of open-channel modification with Ag ϩ supports the suggestion that the structure of the selectivity filter from the bacterial nonselective cation channel is a reasonable model of the selectivity filter of CNG channels (31). Crystal structures determined in the presence of divalent ions show unambiguously that divalent ions bind at the external mouth of the selectivity filter (31), which has been functionally reported in CNG channels (35)(36)(37)(38). If intracellularly applied Ag ϩ is using the selectivity filter to access position T364C, we should expect that external divalent ions would, by electrostatic and/or steric reasons, interfere with the Ag ϩ reaction.…”

Section: Resultssupporting

confidence: 51%

Gating at the selectivity filter in cyclic nucleotide-gated channels

Contreras

Srikumar²,

Holmgren³

2008

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

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By opening and closing the permeation pathway (gating) in response to cGMP binding, cyclic nucleotide-gated (CNG) channels serve key roles in the transduction of visual and olfactory signals. Compiling evidence suggests that the activation gate in CNG channels is not located at the intracellular end of pore, as it has been established for voltage-activated potassium (KV) channels. Here, we show that ion permeation in CNG channels is tightly regulated at the selectivity filter. By scanning the entire selectivity filter using small cysteine reagents, like cadmium and silver, we observed a state-dependent accessibility pattern consistent with gated access at the middle of the selectivity filter, likely at the corresponding position known to regulate structural changes in KcsA channels in response to low concentrations of permeant ions.cGMP ͉ ion channel ͉ signal transduction C yclic nucleotide-gated (CNG) channels sense variations in the intracellular concentration of cyclic nucleotides that occur in response to visual or olfactory stimuli, therefore playing essential roles in the transduction of visual and olfactory information (1, 2). In many ways, CNG channels are similar to voltage-activated potassium (K V ) channels. They coassemble as tetramers of homologous subunits (3-6), each containing six transmembrane segments (TM), a positively charged TM4 and a reentry P region between TM5 and TM6, suggesting that CNG channels belong to the same superfamily of voltage-activated cation channels (7). The main difference is that CNG channels are only weakly voltage-dependent. Instead, they open and close the pore in response to changes in the intracellular concentrations of cGMP or cAMP, a property conferred by the presence of a cyclic nucleotide binding domain at the C terminus of each subunit (8, 9).Our understanding of how CNG channels open and close their pore in response to cyclic nucleotide binding is much less refined than our understanding of how K V channels gate in response to voltage. A large body of evidence, using a variety of approaches, has established that K V channels open and close their permeation pathway at the intracellular end of the pore (10-17). Attempts to extend those ideas to CNG channels have encountered some resistance. For example, studies using intracellularly applied molecules that block the permeation pathway of CNG channels, such as divalent ions (18), tetracaine (19,20), or quaternary ammonium ions (21), have shown that blockade is not state-dependent, as if these molecules can access the pore in both open and closed channels, which is in stark contrast with the blockade properties observed in K V channels (10,11,14,(22)(23)(24). In addition, experiments examining the state dependence of cysteine modification by intracellular application of methanethiosulfonate (MTS) reagents have failed to show dramatic differences between open and closed states in the inner-vestibule region (21,25,26), results that are inconsistent with an intracellular gate in TM6, as shown in K V channels (12,15).Se...

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Section: Resultssupporting

confidence: 51%

Gating at the selectivity filter in cyclic nucleotide-gated channels

Contreras

Srikumar²,

Holmgren³

2008

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

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“…An interesting and physiologically significant property of CNG channels is their ability to conduct Ca 2ϩ in addition to monovalent cations. Functional studies of Ca 2ϩ permeation and blockage in CNG channels have pointed to the presence of multiple Ca 2ϩ binding sites (7,15,19,20). This prompted us to look for evidence of Ca 2ϩ binding elsewhere in the NaK filter in addition to the external site.…”

Section: Resultsmentioning

confidence: 99%

“…This, in turn, leads to an effective blockage of monovalent currents (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Ca 2ϩ blockage from the extracellular side, a property central to the physiological functioning of CNG channels (1), has been extensively studied by using electrophysiological tools and is thought to arise primarily from the involvement of a conserved acidic residue, usually glutamate, in the selectivity filter of the CNG channel (14)(15)(16)(17). Mutating this residue to a neutral amino acid has been shown to drastically decrease external Ca 2ϩ blockage, whereas substituting glutamate with aspartate preserved it.…”

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confidence: 99%

Structural insight into Ca ²⁺ specificity in tetrameric cation channels

Alam

Shi

Jiang

2007

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

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Apparent blockage of monovalent cation currents by the permeating blocker Ca 2؉ is a physiologically essential phenomenon relevant to cyclic nucleotide-gated (CNG) channels. The recently determined crystal structure of a bacterial homolog of CNG channel pores, the NaK channel, revealed a Ca 2؉ binding site at the extracellular entrance to the selectivity filter. This site is not formed by the side-chain carboxylate groups from the conserved acidic residue, Asp-66 in NaK, conventionally thought to directly chelate Ca 2؉ in CNG channels, but rather by the backbone carbonyl groups of residue Gly-67. Here we present a detailed structural analysis of the NaK channel with a focus on Ca 2؉ permeability and blockage. Our results confirm that the Asp-66 residue, although not involved in direct chelation of Ca 2؉ , plays an essential role in external Ca 2؉ binding. Furthermore, we give evidence for the presence of a second Ca 2؉ binding site within the NaK selectivity filter where monovalent cations also bind, providing a structural basis for Ca 2؉ permeation through the NaK pore. Compared with other Ca 2؉ -binding proteins, both sites in NaK present a novel mode of Ca 2؉ chelation, using only backbone carbonyl oxygen atoms from residues in the selectivity filter. The external site is under indirect control by an acidic residue (Asp-66), making it Ca 2؉ -specific. These findings give us a glimpse of the possible underlying mechanisms allowing Ca 2؉ to act both as a permeating ion and blocker of CNG channels and raise the possibility of a similar chemistry governing Ca 2؉ chelation in Ca 2؉ channels.calcium blockage ͉ cyclic nucleotide-gated channel pore ͉ NaK channel ͉ nonselective cation channel

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“…The Ca 2ϩ entry is important because it provides a negative feedback that promotes both recovery from the light response and light adaptation (for review, see Pugh and Lamb, 2000). Because Ca 2ϩ ions pass more slowly through the channel pore than Na ϩ ions, extracellular Ca 2ϩ and Mg 2ϩ block the Na ϩ current by competing for a common intrapore binding site that is formed by glutamate residues of CNGA subunits (Root and MacKinnon, 1993;Eismann et al, 1994;Seifert et al, 1999). We reasoned that the T369S substitution adjacent to Glu-368, the pore glutamate in human CNGA3, may perturb the binding of Ca 2ϩ .…”

Section: Characterization Of Homomeric Wild-type and Mutant Cnga3 Chamentioning

confidence: 99%

Molecular Basis of an Inherited Form of Incomplete Achromatopsia

Tränkner¹,

Jägle²,

Kohl³

et al. 2004

J. Neurosci.

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Mutations in the genes encoding the CNGA3 and CNGB3 subunits of the cyclic nucleotide-gated (CNG) channel of cone photoreceptors have been associated with autosomal recessive achromatopsia. Here we analyze the molecular basis of achromatopsia in two siblings with residual cone function. Psychophysical and electroretinographic analyses show that the light sensitivity of the cone system is lowered, and the signal transfer from cones to secondary neurons is perturbed. Both siblings carry two mutant CNGA3 alleles that give rise to channel subunits with different single-amino acid substitutions. Heterologous expression revealed that only one mutant forms functional channels, albeit with grossly altered properties, including changes in Ca 2ϩ blockage and permeation. Surprisingly, coexpression of this mutant subunit with CNGB3 rescues the channel phenotype, except for the Ca 2ϩ interaction. We argue that these alterations are responsible for the perturbations in light sensitivity and synaptic transmission.

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Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel

Cited by 214 publications

References 21 publications

Gating at the selectivity filter in cyclic nucleotide-gated channels

Gating at the selectivity filter in cyclic nucleotide-gated channels

Structural insight into Ca ²⁺ specificity in tetrameric cation channels

Molecular Basis of an Inherited Form of Incomplete Achromatopsia

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Identification of an external divalent cation-binding site in the pore of a cGMP-activated channel

Cited by 214 publications

References 21 publications

Gating at the selectivity filter in cyclic nucleotide-gated channels

Gating at the selectivity filter in cyclic nucleotide-gated channels

Structural insight into Ca 2+ specificity in tetrameric cation channels

Molecular Basis of an Inherited Form of Incomplete Achromatopsia

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Structural insight into Ca ²⁺ specificity in tetrameric cation channels