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Hot on the trail of TRP channel structure.

Moiseenkova-Bell VY, Wensel TG - J. Gen. Physiol. (2009)

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

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To date, no structural information at the atomic level is available for any full-length TRP channels due to difficulties encountered in overexpression, functional purification, and crystallization of eukaryotic transmembrane proteins... However, progress toward obtaining TRP protein structures has been made by combining different techniques, such as cryo-electron microscopy of entire proteins, x-ray crystallography of isolated cytosolic domains, and extensive mutagenesis combined with functional assays in heterologous expression systems... By analogy to potassium channels related to the Shaker family, the ion pore is predicted to be formed by the combination of the S5 and S6 segments with the “P-loop” connecting them... Within the cytoplasmic domains, some well-known structural motifs have been identified by sequence comparisons: variable numbers of ankyrin repeats (TRPV, TRPA, TRPC, and TRPN), a “TRP” sequence of unknown structure and function found in some but not all family members, kinase domains in TRPM, and extracellular domains inserted into the transmembrane domain in TRPP and TRPML... The cytoplasmic domains of TRP channels make up most of their mass, so solving fragment structures by x-ray crystallography or nuclear magnetic resonance may provide high resolution structures of most of the protein, which could then be fit into lower resolution structures of the full-length proteins determined by electron microscopy (see below)... Another alternative is to use eukaryotic microbes, such as the methylotropic yeast, Pichia pastori, or the budding yeast, Saccharomyces cerevisiae, for expressing TRP channels... The Pichia system has been used successfully for aquaporin and potassium channels, but so far no success has been obtained for TRP channels... Results from TRPM2 and TRPC3 suggest that each is bullet-shaped, with a dense bullet-head domain, interpreted as the transmembrane channel domain, and a more open but larger putative cytoplasmic domain... From the TRPC3 results, the height of the protein was calculated to be 235 Å, and the top view had a width of 200 Å; for TRPM2, the height of the protein reported to be 250 Å, and the top view had a width of 170 Å... One of these is compact and of appropriate volume and length (along the axis of symmetry; presumably the direction of the transmembrane vector) to be the transmembrane domain... This domain measures 40 Å (length) by 60 Å (diameter) and was interpreted as containing the ion channel pore and associated transmembrane helices... The structure of the ankyrin repeat domain of TRPV1, from its N-terminal region, fits well into four shoulder-like domains in the putative cytoplasmic domain near the proposed membrane surface, although this placement must be considered hypothetical until a higher resolution structure is obtained... Preliminary results from TRPV2 suggest its overall structural architecture resembles that of TRPV1 (Moiseenkova-Bell, V., L... Expression systems and purification procedures have been worked out for both full-length proteins and soluble domains, and it is likely that these methods will be extended to additional TRP channels and their domains... The amounts of some full-length TRP channels obtained by expression in budding yeast are sufficient for extensive crystallization trials.

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Structure of TRPV1 at a 19-Å resolution from single-particle analysis and electron cryomicroscopy (Moiseenkova-Bell et al., 2008). Semitransparent orthogonal surface views are shown from (A) the “top” (the side suggested to face the extracellular milieu), (B) the “bottom” (the side suggested to be cytoplasmic), and (C and D) two “side” (perpendicular to the fourfold symmetry axis) directions. In E, a cutaway view is shown to reveal the empty cavity within the basket-like cytoplasmic domain. A bracket indicates the proposed transmembrane domain.
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fig2: Structure of TRPV1 at a 19-Å resolution from single-particle analysis and electron cryomicroscopy (Moiseenkova-Bell et al., 2008). Semitransparent orthogonal surface views are shown from (A) the “top” (the side suggested to face the extracellular milieu), (B) the “bottom” (the side suggested to be cytoplasmic), and (C and D) two “side” (perpendicular to the fourfold symmetry axis) directions. In E, a cutaway view is shown to reveal the empty cavity within the basket-like cytoplasmic domain. A bracket indicates the proposed transmembrane domain.

Mentions: An alternative approach to structure determination by electron microscopy is the use of samples captured without stain or fixative in vitreous ice. Although the limited image contrast obtained using this method presents challenges for proteins <500 kD, the hardware and software available have been improving steadily, so that now structures of noncrystalline specimens at a resolution close to 4 Å can be obtained under favorable conditions (Ludtke et al., 2008). Structures in this resolution range have not yet been determined for TRP channels, but progress has been made in determining lower resolution structures. Recently (Moiseenkova-Bell et al., 2008), electron cryo-microscopy and single-particle analysis were used to determine the structure of TRVP1 to a 19-Å resolution (Fig. 2). The structure is fourfold symmetric and consists of two well-defined domains. One of these is compact and of appropriate volume and length (along the axis of symmetry; presumably the direction of the transmembrane vector) to be the transmembrane domain. This domain measures 40 Å (length) by 60 Å (diameter) and was interpreted as containing the ion channel pore and associated transmembrane helices. The voltage-gated potassium channel Kv 1.2 has a similar six-loop topology in its transmembrane domain to that predicted for TRP channels, and its structure, as determined by x-ray crystallography (Long et al., 2005), fits well into the putative transmembrane domain of the TRPV1 structure. In addition, a large basket-like domain hangs from the transmembrane domain by fairly thin connecting densities. This domain has sufficient volume to contain both the N- and C-terminal cytoplasmic domains. Its overall dimensions are 100 Å (diameter) by 75 Å (length along symmetry axis), but its surface encloses a large central cavity of unknown function. The structure of the ankyrin repeat domain of TRPV1 (Lishko et al., 2007), from its N-terminal region, fits well into four shoulder-like domains in the putative cytoplasmic domain near the proposed membrane surface, although this placement must be considered hypothetical until a higher resolution structure is obtained. Preliminary results from TRPV2 suggest its overall structural architecture resembles that of TRPV1 (Moiseenkova-Bell, V., L. Stanciu, I. Serysheva, B. Tobe, Y. Zhou, and T.G. Wensel. 2007. 51st Annual Biophysical Society Meeting. Abstr. 2626).


Hot on the trail of TRP channel structure.

Moiseenkova-Bell VY, Wensel TG - J. Gen. Physiol. (2009)

Structure of TRPV1 at a 19-Å resolution from single-particle analysis and electron cryomicroscopy (Moiseenkova-Bell et al., 2008). Semitransparent orthogonal surface views are shown from (A) the “top” (the side suggested to face the extracellular milieu), (B) the “bottom” (the side suggested to be cytoplasmic), and (C and D) two “side” (perpendicular to the fourfold symmetry axis) directions. In E, a cutaway view is shown to reveal the empty cavity within the basket-like cytoplasmic domain. A bracket indicates the proposed transmembrane domain.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654091&req=5

fig2: Structure of TRPV1 at a 19-Å resolution from single-particle analysis and electron cryomicroscopy (Moiseenkova-Bell et al., 2008). Semitransparent orthogonal surface views are shown from (A) the “top” (the side suggested to face the extracellular milieu), (B) the “bottom” (the side suggested to be cytoplasmic), and (C and D) two “side” (perpendicular to the fourfold symmetry axis) directions. In E, a cutaway view is shown to reveal the empty cavity within the basket-like cytoplasmic domain. A bracket indicates the proposed transmembrane domain.
Mentions: An alternative approach to structure determination by electron microscopy is the use of samples captured without stain or fixative in vitreous ice. Although the limited image contrast obtained using this method presents challenges for proteins <500 kD, the hardware and software available have been improving steadily, so that now structures of noncrystalline specimens at a resolution close to 4 Å can be obtained under favorable conditions (Ludtke et al., 2008). Structures in this resolution range have not yet been determined for TRP channels, but progress has been made in determining lower resolution structures. Recently (Moiseenkova-Bell et al., 2008), electron cryo-microscopy and single-particle analysis were used to determine the structure of TRVP1 to a 19-Å resolution (Fig. 2). The structure is fourfold symmetric and consists of two well-defined domains. One of these is compact and of appropriate volume and length (along the axis of symmetry; presumably the direction of the transmembrane vector) to be the transmembrane domain. This domain measures 40 Å (length) by 60 Å (diameter) and was interpreted as containing the ion channel pore and associated transmembrane helices. The voltage-gated potassium channel Kv 1.2 has a similar six-loop topology in its transmembrane domain to that predicted for TRP channels, and its structure, as determined by x-ray crystallography (Long et al., 2005), fits well into the putative transmembrane domain of the TRPV1 structure. In addition, a large basket-like domain hangs from the transmembrane domain by fairly thin connecting densities. This domain has sufficient volume to contain both the N- and C-terminal cytoplasmic domains. Its overall dimensions are 100 Å (diameter) by 75 Å (length along symmetry axis), but its surface encloses a large central cavity of unknown function. The structure of the ankyrin repeat domain of TRPV1 (Lishko et al., 2007), from its N-terminal region, fits well into four shoulder-like domains in the putative cytoplasmic domain near the proposed membrane surface, although this placement must be considered hypothetical until a higher resolution structure is obtained. Preliminary results from TRPV2 suggest its overall structural architecture resembles that of TRPV1 (Moiseenkova-Bell, V., L. Stanciu, I. Serysheva, B. Tobe, Y. Zhou, and T.G. Wensel. 2007. 51st Annual Biophysical Society Meeting. Abstr. 2626).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

To date, no structural information at the atomic level is available for any full-length TRP channels due to difficulties encountered in overexpression, functional purification, and crystallization of eukaryotic transmembrane proteins... However, progress toward obtaining TRP protein structures has been made by combining different techniques, such as cryo-electron microscopy of entire proteins, x-ray crystallography of isolated cytosolic domains, and extensive mutagenesis combined with functional assays in heterologous expression systems... By analogy to potassium channels related to the Shaker family, the ion pore is predicted to be formed by the combination of the S5 and S6 segments with the “P-loop” connecting them... Within the cytoplasmic domains, some well-known structural motifs have been identified by sequence comparisons: variable numbers of ankyrin repeats (TRPV, TRPA, TRPC, and TRPN), a “TRP” sequence of unknown structure and function found in some but not all family members, kinase domains in TRPM, and extracellular domains inserted into the transmembrane domain in TRPP and TRPML... The cytoplasmic domains of TRP channels make up most of their mass, so solving fragment structures by x-ray crystallography or nuclear magnetic resonance may provide high resolution structures of most of the protein, which could then be fit into lower resolution structures of the full-length proteins determined by electron microscopy (see below)... Another alternative is to use eukaryotic microbes, such as the methylotropic yeast, Pichia pastori, or the budding yeast, Saccharomyces cerevisiae, for expressing TRP channels... The Pichia system has been used successfully for aquaporin and potassium channels, but so far no success has been obtained for TRP channels... Results from TRPM2 and TRPC3 suggest that each is bullet-shaped, with a dense bullet-head domain, interpreted as the transmembrane channel domain, and a more open but larger putative cytoplasmic domain... From the TRPC3 results, the height of the protein was calculated to be 235 Å, and the top view had a width of 200 Å; for TRPM2, the height of the protein reported to be 250 Å, and the top view had a width of 170 Å... One of these is compact and of appropriate volume and length (along the axis of symmetry; presumably the direction of the transmembrane vector) to be the transmembrane domain... This domain measures 40 Å (length) by 60 Å (diameter) and was interpreted as containing the ion channel pore and associated transmembrane helices... The structure of the ankyrin repeat domain of TRPV1, from its N-terminal region, fits well into four shoulder-like domains in the putative cytoplasmic domain near the proposed membrane surface, although this placement must be considered hypothetical until a higher resolution structure is obtained... Preliminary results from TRPV2 suggest its overall structural architecture resembles that of TRPV1 (Moiseenkova-Bell, V., L... Expression systems and purification procedures have been worked out for both full-length proteins and soluble domains, and it is likely that these methods will be extended to additional TRP channels and their domains... The amounts of some full-length TRP channels obtained by expression in budding yeast are sufficient for extensive crystallization trials.

Show MeSH
Related in: MedlinePlus