Abstract
The sulfonylurea receptor SUR1 associates with Kir6.2 or Kir6.1 to form K(ATP) channels, which link metabolism to excitability in multiple cell types. The strong physical coupling of SUR1 with Kir6 subunits appears exclusive, but recent studies argue that SUR1 also modulates TRPM4, a member of the transient receptor potential family of non-selective cation channels. It has been reported that, following stroke, brain, or spinal cord injury, SUR1 is increased in neurovascular cells at the site of injury. This is accompanied by up-regulation of a non-selective cation conductance with TRPM4-like properties and apparently sensitive to sulfonylureas, leading to the postulation that post-traumatic non-selective cation currents are determined by TRPM4/SUR1 channels. To investigate the mechanistic hypothesis for the coupling between TRPM4 and SUR1, we performed electrophysiological and FRET studies in COSm6 cells expressing TRPM4 channels with or without SUR1. TRPM4-mediated currents were Ca(2+)-activated, voltage-dependent, underwent desensitization, and were inhibited by ATP but were insensitive to glibenclamide and tolbutamide. These properties were not affected by cotransfection with SUR1. When the same SUR1 was cotransfected with Kir6.2, functional K(ATP) channels were formed. In cells cotransfected with Kir6.2, SUR1, and TRPM4, we measured K(ATP)-mediated K(+) currents and Ca(2+)-activated, sulfonylurea-insensitive Na(+) currents in the same patch, further showing that SUR1 controls K(ATP) channel activity but not TRPM4 channels. FRET signal between fluorophore-tagged TRPM4 subunits was similar to that between Kir6.2 and SUR1, whereas there was no detectable FRET efficiency between TRPM4 and SUR1. Our data suggest that functional or structural association of TRPM4 and SUR1 is unlikely.
Highlights
SUR1, the regulatory subunit of KATP channels, was hypothesized to associate with TRPM4 to form novel channels, implicated in cell death following neurovascular trauma
In TRPM4 and TRPM4 ϩ SUR1, currents on cell and in inside-out excised patches in absence of intracellular Ca2ϩ were indistinguishable from those measured in mock-transfected GFP cells (Fig. 2A, bottom panel), e.g. 0.013 Ϯ 0.002 nA on cell, and 0.024 Ϯ 0.003 nA in excised patches at ϩ100 mV (n ϭ 8 each)
In cells cotransfected with TRPM4 and SUR1, Ca2ϩ-activated currents peaked at 2.7 Ϯ 0.7 nA (Fig. 2C; p ϭ 0.43 when compared with TRPM4 cells, unpaired Student’s t test), with on ϭ 2.1 Ϯ 0.4 s (Fig. 2D; p ϭ 0.72)
Summary
SUR1, the regulatory subunit of KATP channels, was hypothesized to associate with TRPM4 to form novel channels, implicated in cell death following neurovascular trauma. There is a dramatic up-regulation of a non-selective cation conductance that has recently been suggested to underlie the brain edema and secondary hemorrhage that often follow and contribute to the devastating effects of stroke, brain, or spinal cord trauma [13] This conductance has been characterized as non-selective for monovalent cations, Ca2ϩ-activated but Ca2ϩ-impermeable, and ATP-sensitive [11, 14], these properties being very reminiscent of those exhibited by TRPM4 channels studied in recombinant cell lines [15, 16]. This conductance appears to be reduced by sulfonylurea drugs such as glibenclamide, and enhanced in the presence of the SUR1-specific KATP
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