Limits...
Multiple unbiased prospective screens identify TRP channels and their conserved gating elements.

Myers BR, Saimi Y, Julius D, Kung C - J. Gen. Physiol. (2008)

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of California, San Francisco, CA 94143, USA.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Each subunit bears extensive N- and C-terminal cytoplasmic domains and an S1-S2-S3-S4-S5-P-S6 motif, where S's are segments of transmembrane α helices and P is the “pore” that houses the ion filter... Indeed, recent electron cryomicroscopy of a TRP channel (TRPV1) at 19-Å resolution is consistent with a tetrameric structure having a compact transmembrane core and a large cytoplasmic domain in the form of a “hanging gondola”... In this way, PKD1 and PKD2 were linked to polycystic kidney disease, founding the TRPP subfamily... Likewise, mucolipidosis type IV was traced to MCOLN1, founding the TRPML subfamily... Indeed, this convergence of discoveries from unbiased independent screens strongly endorses the central role of TRPs in sensory biology as essential components of cellular signaling pathways that sense the environment... Once a TRP channel has been identified from the above searches or by homology with founding members, it is possible to perform site-directed mutageneses to examine the effects of channel mutations, usually by expressing the mutant TRP in oocytes or cultured mammalian cells... Such strategies have been described for the identification of functionally interesting residues or domains in TRPV1, TRPV3, and TRPM8... The founding mutations of TRPs that blind the fly are loss-of-function alleles... The mutants identified in this manner were examined electrophysiologically in Xenopus oocytes... Consistent with the cellular phenotype in yeast, some of the gain-of-function mutants were found to potentiate the effect of acidity, a known gating modality of TRPV1... Note that the mammalian TRPV1 is a heat/inflammation/pain sensor; fungal TRPY1 is a mechanosensitive channel that monitors osmolarity in vivo, whereas Drosophila TRPC1 is a “receptor-operated” channel that functions as a component of the insect phototransduction pathway... That mutations at the same residue near the beginning of S5 were found to have the same molecular effects in three TRPs that play disparate biological roles strongly indicates a common molecular mechanism in the gating of these channels... For example, the F640L mutation of TRPV1 in the predicted pore-helix domain and the F458H mutation of TRPY1 at a point near the predicted hydrophobic occlusion can be expected to have profound effects on channel conformations... Interestingly, the constitutive pore helix mutations recovered from the TRPV1 yeast screen were found to produce similar effects when introduced into TRPV2 and TRPV3, supporting the notion that this region of TRPV channels forms a conserved part of the gating machinery... In summary, assumption-free unbiased searches have pioneered many areas of biological research, a strategy that has now been extended to the analysis of TRP channel physiology.

Show MeSH

Related in: MedlinePlus

An alignment of the S5's (the predicted fifth transmembrane α helices) of different TRP subtypes. In three independent unbiased searches, the same site (red star) was discovered at which mutations cause constitutive channel activities in TRPC1, TRPV1, and TRPY (bold red letters). The site is in a small cluster of phenylalanines, members of which are found in all TRP subtypes (underlined red). The mutations in TRPV4 that cause brachyolmia in human (green) and the mutation in TRPML that causes the varitint-waddler mouse phenotype are nearby (orange). Shown are subfamily representatives: TRPA (painless of Drosophila), TRPC (the canonical TRPC of Drosophila), TRPM2 (human), TRPML3 (mouse), TRPN (zebra fish), TRPP2 (mouse), TRPV1 (rat), TRPV4 (rat), and TRPY1 (budding yeast). Analyses began with a large-scale alignment of the full-length sequences of all current members of the TRP superfamily, along with several other 6-S cation channels, using the CLUSTAL W algorithm (Gonnet 250 matrix) by way of the CLUSTAL X interface (1.81) (Thompson et al., 1997). Comparisons with pfam00520 (Finn et al., 2006) and applications of various transmembrane and secondary-structure prediction algorithms (e.g., PROFphd; Thompson et al., 1997; Rost et al., 2004; Finn et al., 2006) predict the sequences shown here to be the transmembrane α helices preceding the pore helix.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2571970&req=5

fig1: An alignment of the S5's (the predicted fifth transmembrane α helices) of different TRP subtypes. In three independent unbiased searches, the same site (red star) was discovered at which mutations cause constitutive channel activities in TRPC1, TRPV1, and TRPY (bold red letters). The site is in a small cluster of phenylalanines, members of which are found in all TRP subtypes (underlined red). The mutations in TRPV4 that cause brachyolmia in human (green) and the mutation in TRPML that causes the varitint-waddler mouse phenotype are nearby (orange). Shown are subfamily representatives: TRPA (painless of Drosophila), TRPC (the canonical TRPC of Drosophila), TRPM2 (human), TRPML3 (mouse), TRPN (zebra fish), TRPP2 (mouse), TRPV1 (rat), TRPV4 (rat), and TRPY1 (budding yeast). Analyses began with a large-scale alignment of the full-length sequences of all current members of the TRP superfamily, along with several other 6-S cation channels, using the CLUSTAL W algorithm (Gonnet 250 matrix) by way of the CLUSTAL X interface (1.81) (Thompson et al., 1997). Comparisons with pfam00520 (Finn et al., 2006) and applications of various transmembrane and secondary-structure prediction algorithms (e.g., PROFphd; Thompson et al., 1997; Rost et al., 2004; Finn et al., 2006) predict the sequences shown here to be the transmembrane α helices preceding the pore helix.

Mentions: The founding mutations of TRPs that blind the fly are loss-of-function alleles. As noted above, the inability to sustain the light-induced receptor potential results from loss of function for the Drosophila TRPC1 channel, originally called trp (Cosens and Manning, 1969; Minke et al., 1975; Montell and Rubin, 1989). A lone gain-of-function dTRPC1 allele was later identified, which results not only in blindness, but also rapid and severe retinal degeneration (Yoon et al., 2000). In this fly, receptor potential is sustained not only during the light presentation, but long after the light is turned off. The retinal degeneration is presumably caused by the cytotoxicity of Ca2+ entering through the constitutively active channels. The causative mutation was identified as F550I (Fig. 1, red), located near the beginning of the predicted S5 (Hong et al., 2002), within a cluster of three phenylalanines. In fact, one or more phenylalanines can be found in this region of members of fungal and animal TRP subfamilies (Fig. 1, underlined).


Multiple unbiased prospective screens identify TRP channels and their conserved gating elements.

Myers BR, Saimi Y, Julius D, Kung C - J. Gen. Physiol. (2008)

An alignment of the S5's (the predicted fifth transmembrane α helices) of different TRP subtypes. In three independent unbiased searches, the same site (red star) was discovered at which mutations cause constitutive channel activities in TRPC1, TRPV1, and TRPY (bold red letters). The site is in a small cluster of phenylalanines, members of which are found in all TRP subtypes (underlined red). The mutations in TRPV4 that cause brachyolmia in human (green) and the mutation in TRPML that causes the varitint-waddler mouse phenotype are nearby (orange). Shown are subfamily representatives: TRPA (painless of Drosophila), TRPC (the canonical TRPC of Drosophila), TRPM2 (human), TRPML3 (mouse), TRPN (zebra fish), TRPP2 (mouse), TRPV1 (rat), TRPV4 (rat), and TRPY1 (budding yeast). Analyses began with a large-scale alignment of the full-length sequences of all current members of the TRP superfamily, along with several other 6-S cation channels, using the CLUSTAL W algorithm (Gonnet 250 matrix) by way of the CLUSTAL X interface (1.81) (Thompson et al., 1997). Comparisons with pfam00520 (Finn et al., 2006) and applications of various transmembrane and secondary-structure prediction algorithms (e.g., PROFphd; Thompson et al., 1997; Rost et al., 2004; Finn et al., 2006) predict the sequences shown here to be the transmembrane α helices preceding the pore helix.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571970&req=5

fig1: An alignment of the S5's (the predicted fifth transmembrane α helices) of different TRP subtypes. In three independent unbiased searches, the same site (red star) was discovered at which mutations cause constitutive channel activities in TRPC1, TRPV1, and TRPY (bold red letters). The site is in a small cluster of phenylalanines, members of which are found in all TRP subtypes (underlined red). The mutations in TRPV4 that cause brachyolmia in human (green) and the mutation in TRPML that causes the varitint-waddler mouse phenotype are nearby (orange). Shown are subfamily representatives: TRPA (painless of Drosophila), TRPC (the canonical TRPC of Drosophila), TRPM2 (human), TRPML3 (mouse), TRPN (zebra fish), TRPP2 (mouse), TRPV1 (rat), TRPV4 (rat), and TRPY1 (budding yeast). Analyses began with a large-scale alignment of the full-length sequences of all current members of the TRP superfamily, along with several other 6-S cation channels, using the CLUSTAL W algorithm (Gonnet 250 matrix) by way of the CLUSTAL X interface (1.81) (Thompson et al., 1997). Comparisons with pfam00520 (Finn et al., 2006) and applications of various transmembrane and secondary-structure prediction algorithms (e.g., PROFphd; Thompson et al., 1997; Rost et al., 2004; Finn et al., 2006) predict the sequences shown here to be the transmembrane α helices preceding the pore helix.
Mentions: The founding mutations of TRPs that blind the fly are loss-of-function alleles. As noted above, the inability to sustain the light-induced receptor potential results from loss of function for the Drosophila TRPC1 channel, originally called trp (Cosens and Manning, 1969; Minke et al., 1975; Montell and Rubin, 1989). A lone gain-of-function dTRPC1 allele was later identified, which results not only in blindness, but also rapid and severe retinal degeneration (Yoon et al., 2000). In this fly, receptor potential is sustained not only during the light presentation, but long after the light is turned off. The retinal degeneration is presumably caused by the cytotoxicity of Ca2+ entering through the constitutively active channels. The causative mutation was identified as F550I (Fig. 1, red), located near the beginning of the predicted S5 (Hong et al., 2002), within a cluster of three phenylalanines. In fact, one or more phenylalanines can be found in this region of members of fungal and animal TRP subfamilies (Fig. 1, underlined).

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of California, San Francisco, CA 94143, USA.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Each subunit bears extensive N- and C-terminal cytoplasmic domains and an S1-S2-S3-S4-S5-P-S6 motif, where S's are segments of transmembrane α helices and P is the “pore” that houses the ion filter... Indeed, recent electron cryomicroscopy of a TRP channel (TRPV1) at 19-Å resolution is consistent with a tetrameric structure having a compact transmembrane core and a large cytoplasmic domain in the form of a “hanging gondola”... In this way, PKD1 and PKD2 were linked to polycystic kidney disease, founding the TRPP subfamily... Likewise, mucolipidosis type IV was traced to MCOLN1, founding the TRPML subfamily... Indeed, this convergence of discoveries from unbiased independent screens strongly endorses the central role of TRPs in sensory biology as essential components of cellular signaling pathways that sense the environment... Once a TRP channel has been identified from the above searches or by homology with founding members, it is possible to perform site-directed mutageneses to examine the effects of channel mutations, usually by expressing the mutant TRP in oocytes or cultured mammalian cells... Such strategies have been described for the identification of functionally interesting residues or domains in TRPV1, TRPV3, and TRPM8... The founding mutations of TRPs that blind the fly are loss-of-function alleles... The mutants identified in this manner were examined electrophysiologically in Xenopus oocytes... Consistent with the cellular phenotype in yeast, some of the gain-of-function mutants were found to potentiate the effect of acidity, a known gating modality of TRPV1... Note that the mammalian TRPV1 is a heat/inflammation/pain sensor; fungal TRPY1 is a mechanosensitive channel that monitors osmolarity in vivo, whereas Drosophila TRPC1 is a “receptor-operated” channel that functions as a component of the insect phototransduction pathway... That mutations at the same residue near the beginning of S5 were found to have the same molecular effects in three TRPs that play disparate biological roles strongly indicates a common molecular mechanism in the gating of these channels... For example, the F640L mutation of TRPV1 in the predicted pore-helix domain and the F458H mutation of TRPY1 at a point near the predicted hydrophobic occlusion can be expected to have profound effects on channel conformations... Interestingly, the constitutive pore helix mutations recovered from the TRPV1 yeast screen were found to produce similar effects when introduced into TRPV2 and TRPV3, supporting the notion that this region of TRPV channels forms a conserved part of the gating machinery... In summary, assumption-free unbiased searches have pioneered many areas of biological research, a strategy that has now been extended to the analysis of TRP channel physiology.

Show MeSH
Related in: MedlinePlus