Structural themes in ion channels

Doyle, D.A.

doi:10.1007/s00249-003-0382-z

Cited by 26 publications

(14 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6C, n ϭ 5, p Ͻ 0.001) is consistent with the notion that Glu-316 and Glu-320 play a role in concentrating cations near the periplasmic pore entrance (5,16). Thus, CorA appears to utilize at least one type of electrostatic sink, a property that it shares with numerous other ion channel and transporter proteins (46).…”

Section: Structure-function Relationships In Tmcorasupporting

confidence: 80%

Probing Structure-Function Relationships and Gating Mechanisms in the CorA Mg2+ Transport System

Payandeh

Ramjeesingh

et al. 2008

Journal of Biological Chemistry

102

View full text Add to dashboard Cite

Recent crystal structures of the CorA Mg2؉ transport protein from Thermotoga maritima (TmCorA) revealed an unusually long ion pore putatively gated by hydrophobic residues near the intracellular end and by universally conserved asparagine residues at the periplasmic entrance. A conformational change observed in an isolated funnel domain structure also led to a proposal for the structural basis of gating. Because understanding the molecular mechanisms underlying ion channel and transporter gating remains an important challenge, we have undertaken a structure-guided engineering approach to probe structure-function relationships in TmCorA. The intracellular funnel domain is shown to constitute an allosteric regulatory module that can be engineered to promote an activated or closed state. A periplasmic gate centered about a proline-induced kink of the pore-lining helix is described where "helix-straightening" mutations produce a dramatic gain-of-function. Mutation to the narrowest constriction along the pore demonstrates that a hydrophobic gate is operational within this Mg 2؉ -selective transport protein and likely forms an energetic barrier to ion flux. We also provide evidence that highly conserved acidic residues found in the short periplasmic loop are not essential for TmCorA function or Mg 2؉ selectivity but may be required for proper protein folding and stability. This work extends our gating model for the CorA-Alr1-Mrs2 superfamily and reveals features that are characteristic of an ion channel. Aspects of these results that have broader implications for a range of channel and transporter families are highlighted.The magnesium ion is essential for life and performs crucial roles in biology. As the major intracellular divalent cation,

show abstract

Section: Structure-function Relationships In Tmcorasupporting

confidence: 80%

Probing Structure-Function Relationships and Gating Mechanisms in the CorA Mg2+ Transport System

Payandeh

Ramjeesingh

et al. 2008

Journal of Biological Chemistry

102

View full text Add to dashboard Cite

show abstract

“…Together with the analyses of results from mutational studies it has been concluded that K + symporter families from prokaryotes and eukaryotes have evolved from the prokaryotic K + channel proteins (Durell et al, 1999;Durell & Guy, 2001). The findings were used to clone and overexpress a few additional bacterial proteins (as done for KcsA) and to generate their crystal structure in order to understand the general principle of ion channels (see review by Doyle, 2004). The results of experimental data (Meuser et al, 2001) support the conclusion that the crystal structure of the tetrameric KcsA does not present the 'open state', as initially concluded (Doyle et al, 1998).…”

Section: Introductionmentioning

confidence: 73%

In vivo monitoring of the potassium channel KcsA in Streptomyces lividans hyphae using immuno-electron microscopy and energy-filtering transmission electron microscopy

2006

View full text Add to dashboard Cite

The previous discovery of the Streptomyces lividans kcsA gene and its overexpression followed by the functional reconstitution of the purified gene product has resulted in new strategies to explore this channel protein in vitro. KcsA has evolved as a general model to investigate the structure/ function relationship of ion channel proteins. Using specific antibodies raised against a domain of KcsA lacking membrane-spanning regions, KcsA has now been localized within numerous separated clusters between the outer face of the cytoplasm and the cell envelope in substrate hyphae of the S. lividans wild-type strain but not in a designed chromosomal disruption mutant DK, lacking a functional kcsA gene. Previous findings had revealed that caesium ions led to a block of KcsA channel activity within S. lividans protoplasts fused to giant vesicles. As caesium can be scored by electron energy loss spectroscopy better than potassium, this technique was applied to hyphae that had been briefly exposed to caesium instead of potassium ions. Caesium was found preferentially at the cell envelope. Compared to the DK mutant, the relative level of caesium was <30 % enhanced in the wild-type. This is attributed to the presence of KcsA channels. Additional visualization by electron spectroscopic imaging supported this conclusion. The data presented are believed to represent the first demonstration of in vivo monitoring of KcsA in its original host.

show abstract

“…One such implication is that the ion transport sites of NKCC, although distinct kinetically, may be capable of interacting with one and another through certain key residues. Indeed, the structural models of the tm2-cs1a region also predicts that residues 216 and 220 (Ile and Ile in saA and Val and Leu in saF) are adjacent to one another on the same vertical axis of the ␣-helix, suggesting that their peptide backbones and/or aliphatic R groups are connected through H atoms (35,36). In the case of NKCC, for which ions are proposed to move single file down a translocation pocket (19 -21, 24, 25), such physical interactions could represent a key aspect of the carrier's modus operandi, accounting, in particular, for the kinetic interdependence of the ion binding sites.…”

Section: Discussionmentioning

confidence: 99%

Molecular Mechanisms of Cation Transport by the Renal Na+-K+-Cl- Cotransporter

Gagnon

Bergeron

Daigle

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

Two variants of the renal Na؉ -K ؉ -Cl ؊ cotransporter (NKCC2), called NKCC2A and NKCC2F, display marked differences in Na ؉ , Rb ؉ , and Cl ؊ affinities, yet are identical to one another except for a 23-residue membrane-associated domain that is derived from alternatively spliced exons. The proximal portion of these exons is predicted to encode the second transmembrane domain (tm2) in the form of an ␣-helix, and the distal portion, part of the following connecting segment (cs1a). In recent studies, we have taken advantage of the A-F differences in kinetic behavior to determine which regions in tm2-cs1a are involved in ion transport. Functional characterizations of chimeras in which tm2 or cs1a were interchanged between the variants showed that both regions are important in specifying ion affinities, but did not allow delineating the contribution of individual residues. Here, we have extended these structurefunction analyses by studying additional mutants in which variant residues between A and F were interchanged individually in the tm2-cs1a region (amino acid number 216, 220, 223, 229, or 233 in NKCC2). None of the substitutions were found to affect K m (C1؊) , suggesting that the affinity difference for anion transport is conveyed by a combination of variant residues in this domain. However, 2 substitutions in the tm2 of F were found to affect cation constants specifically; interestingly, one of these mutations (residue 216) only affected K m (Rb؉) while the other (residue 220) only affected K m (Na؉) . We have thus identified two novel residues in NKCC2 that play a key role in cation transport. Because such residues should be adjacent to one another on the vertical axis of the tm2 ␣-helix, our results imply, furthermore, that the ion transport sites in NKCC2 could be physically linked.

show abstract

Structural themes in ion channels

Cited by 26 publications

References 25 publications

Probing Structure-Function Relationships and Gating Mechanisms in the CorA Mg2+ Transport System

Probing Structure-Function Relationships and Gating Mechanisms in the CorA Mg2+ Transport System

In vivo monitoring of the potassium channel KcsA in Streptomyces lividans hyphae using immuno-electron microscopy and energy-filtering transmission electron microscopy

Molecular Mechanisms of Cation Transport by the Renal Na+-K+-Cl- Cotransporter

Contact Info

Product

Resources

About