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Identification of pore residues engaged in determining divalent cationic permeation in transient receptor potential melastatin subtype channel 2.

Xia R, Mei ZZ, Mao HJ, Yang W, Dong L, Bradley H, Beech DJ, Jiang LH - J. Biol. Chem. (2008)

Bottom Line: The D987E mutant was functional and showed greater Ca(2+) permeability along with concentration-dependent inhibition of Na(+)-carrying currents by Ca(2+).Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca(2+) permeability.These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.

View Article: PubMed Central - PubMed

Affiliation: Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT
The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca(2+) and Mg(2+) permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg(2+) permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca(2+) permeability along with concentration-dependent inhibition of Na(+)-carrying currents by Ca(2+). Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca(2+)-permeable TRPM channels, substantially increased Ca(2+) permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca(2+) permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.

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Construction and expression of TRPM2 subunit concatemers. A, schematic diagram of TRPM2 subunit concatenation. The linker consists of serine and arginine (SR) residues. Position 960 was glutamate (WT), glutamine (E960Q mutant), or aspartate (E960D mutant), and position 987 was aspartate (WT), asparagine (D987N mutant), or glutamate (D987E mutant). N, N terminus; C, C terminus. B, Western blotting analysis of protein expression of the indicated subunits and subunit concatemers. The proteins were immunoprecipitated and detected by an anti-EE antibody. The arrowheads denote the expected monomeric (M) and dimeric proteins (D), respectively. C, representative ADPR-evoked currents at –80 mV (denoted by circles, top) and I/V curves (bottom), obtained by voltage ramps applied every 5 s, from cells expressing the indicated subunit concatemers. ACA, anthranilic acid. D, summary of the ADPR-evoked peak currents in cells expressing the indicated subunit concatemers. The number of cells examined in each case is indicated. ***, p < 0.001, when compared with WT-WT.
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fig4: Construction and expression of TRPM2 subunit concatemers. A, schematic diagram of TRPM2 subunit concatenation. The linker consists of serine and arginine (SR) residues. Position 960 was glutamate (WT), glutamine (E960Q mutant), or aspartate (E960D mutant), and position 987 was aspartate (WT), asparagine (D987N mutant), or glutamate (D987E mutant). N, N terminus; C, C terminus. B, Western blotting analysis of protein expression of the indicated subunits and subunit concatemers. The proteins were immunoprecipitated and detected by an anti-EE antibody. The arrowheads denote the expected monomeric (M) and dimeric proteins (D), respectively. C, representative ADPR-evoked currents at –80 mV (denoted by circles, top) and I/V curves (bottom), obtained by voltage ramps applied every 5 s, from cells expressing the indicated subunit concatemers. ACA, anthranilic acid. D, summary of the ADPR-evoked peak currents in cells expressing the indicated subunit concatemers. The number of cells examined in each case is indicated. ***, p < 0.001, when compared with WT-WT.

Mentions: Constructs, Cell Culture, and Transfection—The construct encoding human TRPM2 (8) with a C-terminal EE epitope (29) was used. Mutations were introduced using QuikChange system (Stratagene) and confirmed by sequencing. The constructs encoding subunit concatemers with a C-terminal EE epitope were made as follows. Firstly, the sequence (nucleotides 1–700) encoding part of the TRPM2 N terminus (TRPM2N) was amplified by PCR using Pfu and forward primer 5′-TCTCTAGAATGGAGCCCTCAGCCCTGAGG-3′ (XbaI sequence underlined and TRPM2 sequence in italic) and reverse primer 5′-TCAGTACAGGTAGAGCAAGGTGTCC-3′. The resultant PCR product containing XbaI site was inserted into pCR2.1 following the manufacturer's instructions (Invitrogen) to generate TRPM2N-pCR2.1. Secondly, the vector sequence between EcoRI and XbaI and the TRPM2 sequence between XbaI and SacI were separately excised from TRPM2N-pCR2.1 and ligated with the sequence between SacI and EcoRI from TRPM2-EE-pcDNA3.1 to generate TRPM2-EE-pCR2.1. Finally, the TRPM2-EE sequence between XbaI and HindIII was excised from TRPM2-EE-pCR2.1 to replace the sequence between XbaI and PmeI in TRPM2-Myc-pcDNA3.1 to produce the constructs encoding concatenated subunits (see Fig. 4A). Maintenance of human embryonic kidney cells (HEK293) and transient transfection with plasmids were described previously (29).


Identification of pore residues engaged in determining divalent cationic permeation in transient receptor potential melastatin subtype channel 2.

Xia R, Mei ZZ, Mao HJ, Yang W, Dong L, Bradley H, Beech DJ, Jiang LH - J. Biol. Chem. (2008)

Construction and expression of TRPM2 subunit concatemers. A, schematic diagram of TRPM2 subunit concatenation. The linker consists of serine and arginine (SR) residues. Position 960 was glutamate (WT), glutamine (E960Q mutant), or aspartate (E960D mutant), and position 987 was aspartate (WT), asparagine (D987N mutant), or glutamate (D987E mutant). N, N terminus; C, C terminus. B, Western blotting analysis of protein expression of the indicated subunits and subunit concatemers. The proteins were immunoprecipitated and detected by an anti-EE antibody. The arrowheads denote the expected monomeric (M) and dimeric proteins (D), respectively. C, representative ADPR-evoked currents at –80 mV (denoted by circles, top) and I/V curves (bottom), obtained by voltage ramps applied every 5 s, from cells expressing the indicated subunit concatemers. ACA, anthranilic acid. D, summary of the ADPR-evoked peak currents in cells expressing the indicated subunit concatemers. The number of cells examined in each case is indicated. ***, p < 0.001, when compared with WT-WT.
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Related In: Results  -  Collection

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fig4: Construction and expression of TRPM2 subunit concatemers. A, schematic diagram of TRPM2 subunit concatenation. The linker consists of serine and arginine (SR) residues. Position 960 was glutamate (WT), glutamine (E960Q mutant), or aspartate (E960D mutant), and position 987 was aspartate (WT), asparagine (D987N mutant), or glutamate (D987E mutant). N, N terminus; C, C terminus. B, Western blotting analysis of protein expression of the indicated subunits and subunit concatemers. The proteins were immunoprecipitated and detected by an anti-EE antibody. The arrowheads denote the expected monomeric (M) and dimeric proteins (D), respectively. C, representative ADPR-evoked currents at –80 mV (denoted by circles, top) and I/V curves (bottom), obtained by voltage ramps applied every 5 s, from cells expressing the indicated subunit concatemers. ACA, anthranilic acid. D, summary of the ADPR-evoked peak currents in cells expressing the indicated subunit concatemers. The number of cells examined in each case is indicated. ***, p < 0.001, when compared with WT-WT.
Mentions: Constructs, Cell Culture, and Transfection—The construct encoding human TRPM2 (8) with a C-terminal EE epitope (29) was used. Mutations were introduced using QuikChange system (Stratagene) and confirmed by sequencing. The constructs encoding subunit concatemers with a C-terminal EE epitope were made as follows. Firstly, the sequence (nucleotides 1–700) encoding part of the TRPM2 N terminus (TRPM2N) was amplified by PCR using Pfu and forward primer 5′-TCTCTAGAATGGAGCCCTCAGCCCTGAGG-3′ (XbaI sequence underlined and TRPM2 sequence in italic) and reverse primer 5′-TCAGTACAGGTAGAGCAAGGTGTCC-3′. The resultant PCR product containing XbaI site was inserted into pCR2.1 following the manufacturer's instructions (Invitrogen) to generate TRPM2N-pCR2.1. Secondly, the vector sequence between EcoRI and XbaI and the TRPM2 sequence between XbaI and SacI were separately excised from TRPM2N-pCR2.1 and ligated with the sequence between SacI and EcoRI from TRPM2-EE-pcDNA3.1 to generate TRPM2-EE-pCR2.1. Finally, the TRPM2-EE sequence between XbaI and HindIII was excised from TRPM2-EE-pCR2.1 to replace the sequence between XbaI and PmeI in TRPM2-Myc-pcDNA3.1 to produce the constructs encoding concatenated subunits (see Fig. 4A). Maintenance of human embryonic kidney cells (HEK293) and transient transfection with plasmids were described previously (29).

Bottom Line: The D987E mutant was functional and showed greater Ca(2+) permeability along with concentration-dependent inhibition of Na(+)-carrying currents by Ca(2+).Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca(2+) permeability.These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.

View Article: PubMed Central - PubMed

Affiliation: Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT
The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca(2+) and Mg(2+) permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg(2+) permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca(2+) permeability along with concentration-dependent inhibition of Na(+)-carrying currents by Ca(2+). Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca(2+)-permeable TRPM channels, substantially increased Ca(2+) permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca(2+) permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.

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