Identification of Pore Residues Engaged in Determining Divalent Cationic Permeation in Transient Receptor Potential Melastatin Subtype Channel 2

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.The melastatin subtype of transient receptor potential (TRPM) 6 ion channels is widely expressed in neuronal, cardiovascular, immune, and endothelial cells where they are engaged in diverse physiological and pathophysiological processes (1-7). TRPM2 channels are activated by adenosine diphosphoribose (ADPR) and also by oxidative stress and mediate immune function, insulin secretion, endothelial permeability, and cell death that are induced by oxidative stress (8 -14).All members of the transient receptor potential (TRP) channel superfamily, which includes TRPC, TRPV, TRPM, TRPP, TRPML, and TRPA subfamilies, have a basic architecture similar to voltage-gated potassium channels, with homo-or hetero-tetrameric arrangements around a central ion-conducting pore (1,7,15). Each subunit is considered to have intracellular N and C termini and six transmembrane segments (S1-S6) with a re-entrant pore loop connecting S5 and S6 (see Fig. 1A). Despite significant differences in the amino acid residue sequences of the pore loop among different TRP subfamilies, there are two stretches of amino acid residues that are thought to form the pore helix and the ion selectivity filter of these channels, respectively (15).TRPM channels, including TRPM2, show considerable permeability to Ca 2ϩ and other divalent cations, with the exception of TRPM4/5 channels that are selective for monovalent cations (8, 9, 13-18). The molecular basis for divalent cationic permeability of TRPM channels is not fully understood. Accumulating evidence supports an important role of the ion selectivity filter in Ca 2ϩ and Mg 2ϩ permeation of TRPV and TRPM channels (15, 1...

Depletion of the Human Ion Channel TRPM2 in Neuroblastoma Demonstrates Its Key Role in Cell Survival through Modulation of Mitochondrial Reactive Oxygen Species and Bioenergetics

“…The E960D construct was previously generated by our laboratory and the loss of function has been authenticated (38,45). Expression of wild type TRPM2 but not E960D in the KO reconstituted cell viability at or close to the level observed in the scrambled control (Fig.…”

Zinc Inactivates Melastatin Transient Receptor Potential 2 Channels via the Outer Pore

“…2-4). The constructs used were generated in previous studies (61)(62)(63)(64). Chemicals and reagents used were purchased from Sigma unless otherwise indicated.…”

“…Electrophysiology-Patch clamp recordings were made at room temperature as described previously (61)(62)(63)(64), using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). The extracellular solution contained (in mM): 147 NaCl, 2 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 HEPES, and 13 glucose, pH 7.4.…”

Residues 155 and 348 Contribute to the Determination of P2X7 Receptor Function via Distinct Mechanisms Revealed by Single-nucleotide Polymorphisms

“…Biotin Labeling and Western Blotting-Biotin labeling was performed by modifying the protocols described previously (62,63). In brief, cells transfected with P2X 7 plasmid (or an empty vector) and GFP plasmid were labeled with sulfo-NHS-LC-biotin (Pierce) for 30 min at 4°C.…”