Biomedical subjects

Stephen G Aller

Publications and source records attributed to Stephen G Aller.

6 recordsLinked to original sources

PubMed2007

A structural perspective on copper uptake in eukaryotes.

Over a decade ago, genetic studies identified a family of small integral membrane proteins, commonly referred to as copper transporters (CTRs) that are both required and sufficient for cellular copper uptake in a yeast genetic complementation assay. We recently used electron crystallography to determine a projection density map of the human high affinity transporter hCTR1 embedded into a lipid bilayer. At 6 A resolution, this first glimpse of the structure revealed that hCTR1 is trimeric and possesses the type of radial symmetry that traditionally has been associated with the structure of certain ion channels such as potassium or gap junction channels. Representative for this particular type of architecture, a region of low protein density at the center of the trimer is consistent with the existence of a copper permeable pore along the center three-fold axis of the trimer. In this contribution, we will briefly discuss how recent structure-function studies correlate with the projection density map, and provide a perspective with respect to the cellular uptake of other transition metals.

Amino Acid Sequence↗

PubMed2006

Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride.

In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action.

1-Methyl-3-isobutylxanthine↗

PubMed2006

Projection structure of the human copper transporter CTR1 at 6-A resolution reveals a compact trimer with a novel channel-like architecture.

Human CTR1 is a high-affinity copper transporter that also mediates the uptake of the anticancer drug cisplatin by largely unknown transport mechanisms. Here we report the 6-A projection structure obtained for human CTR1 by using electron crystallography of 2D protein crystals in a native phospholipid bilayer. The projection of CTR1 reveals a symmetrical trimer that is <40 A wide. Notably, the center threefold axis of each trimer forms a region of very low electron density likely to be involved in copper translocation. The formation of a putative pore for metal ions at the interface of three identical subunits deviates from the structural design of typical primary and secondary active transporters and reveals that copper uptake transporters have a novel architecture that is structurally more closely related to channel proteins.

Antineoplastic Agents↗

PubMed2005

Mercury and zinc differentially inhibit shark and human CFTR orthologues: involvement of shark cysteine 102.

The apical membrane is an important site of mercury toxicity in shark rectal gland tubular cells. We compared the effects of mercury and other thiol-reacting agents on shark CFTR (sCFTR) and human CFTR (hCFTR) chloride channels using two-electrode voltage clamping of cRNA microinjected Xenopus laevis oocytes. Chloride conductance was stimulated by perfusing with 10 microM forskolin (FOR) and 1 mM IBMX, and then thio-reactive species were added. In oocytes expressing sCFTR, FOR + IBMX mean stimulated Cl(-) conductance was inhibited 69% by 1 microM mercuric chloride and 78% by 5 microM mercuric chloride (IC(50) of 0.8 microM). Despite comparable stimulation of conductance, hCFTR was insensitive to 1 microM HgCl(2) and maximum inhibition was 15% at the highest concentration used (5 microM). Subsequent exposure to glutathione (GSH) did not reverse the inhibition of sCFTR by mercury, but dithiothreitol (DTT) completely reversed this inhibition. Zinc (50-200 microM) also reversibly inhibited sCFTR (40-75%) but did not significantly inhibit hCFTR. Similar inhibition of sCFTR but not hCFTR was observed with an organic mercurial, p-chloromercuriphenylsulfonic acid (pCMBS). The first membrane spanning domain (MSD1) of sCFTR contains two unique cysteines, C102 and C303. A chimeric construct replacing MSD1 of hCFTR with the corresponding sequence of sCFTR was highly sensitive to mercury. Site-specific mutations introducing the first but not the second shark unique cysteine in hCFTR MSD1 resulted in full sensitivity to mercury. These experiments demonstrate a profound difference in the sensitivity of shark vs. human CFTR to inhibition by three thiol-reactive substances, an effect that involves C102 in the shark orthologue.

4-Chloromercuribenzenesulfonate↗

PubMed2004

Eukaryotic CTR copper uptake transporters require two faces of the third transmembrane domain for helix packing, oligomerization, and function.

Members of the copper uptake transporter (CTR) family from yeast, plants, and mammals including human are required for cellular uptake of the essential metal copper. Based on biochemical data, CTRs have three transmembrane domains and have been shown to oligomerize in the membrane. Among individual members of the family, there is little amino acid sequence identity, raising questions as to how these proteins adopt a common fold, oligomerize, and participate in copper transport. Using site-directed mutagenesis, tryptophan scanning, genetic complementation, subcellular localization, chemical cross-linking, and the yeast unfolded protein response, we demonstrated that at least half of the third transmembrane domain (TM3) plays a vital role in CTR structure and function. The results of our analysis showed that TM3 contains two functionally distinct faces. One face bears a highly conserved Gly-X-X-X-Gly (GG4) motif, which we showed to be essential for CTR oligomerization. Moreover, we showed that steric constraints reach past the GG4-motif itself including amino acid residues that are not conserved throughout the CTR family. A second face of TM3 contains three amino acid positions that, when mutated to tryptophan, cause predominantly abnormal localization but are still partially functional in growth complementation experiments. These mutations cluster on the face opposite to the GG4-bearing face of TM3 where they may mediate interactions with the remaining two transmembrane domains. Taken together, our data support TM3 as being buried within trimeric CTR where it plays an essential role in CTR assembly.

Amino Acid Motifs↗

PubMed2002

The membrane protein FeoB contains an intramolecular G protein essential for Fe(II) uptake in bacteria.

G proteins are critical for the regulation of membrane protein function and signal transduction. Nevertheless, coupling between G proteins and membrane proteins with multiple membrane-spanning domains has so far been observed only in higher organisms. Here we show that the polytopic membrane protein FeoB, which is essential for Fe(II) uptake in bacteria, contains a guanine-nucleotide-specific nucleotide binding site. We identify the G4-motif, NXXD, responsible for guanine nucleotide specificity, and show that GTP hydrolysis occurs very slowly. In contrast to typical G proteins, the association and dissociation of GDP were found to be faster than for GTP, suggesting that in the absence of additional factors, FeoB's G protein domain may exist mostly in the GTP-bound form. Furthermore, the binding of GTP is required for efficient Fe(II) uptake through the FeoB-dependent system. Notably, even in bacteria, this covalent linkage between a G protein and a polytopic membrane protein appears, to our knowledge, to be unique. These findings raise the intriguing question whether FeoB represents a primordial archetype of G protein-regulated membrane proteins.

Adenosine Triphosphate↗