Identification of pore residues engaged in determining divalent cationic permeation in transient receptor potential melastatin subtype channel 2.
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.
Xia
Rong
R
Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Mei
Zhu-Zhong
ZZ
Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Mao
Hong-Ju
HJ
Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Yang
Wei
W
Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Dong
Li
L
Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Bradley
Helen
H
Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Beech
David J
DJ
Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Jiang
Lin-Hua
LH
Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom. Electronic address: l.h.jiang@leeds.ac.uk.
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D002460:Cell Line; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D008274:Magnesium / Q000378:metabolism*; D020125:Mutation, Missense; D010539:Permeability; D012964:Sodium / Q000378:metabolism; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism*
D002413:Cations, Divalent; D050053:TRPM Cation Channels; D001224:Aspartic Acid; D018698:Glutamic Acid; D012964:Sodium; D008274:Magnesium; D002118:Calcium