One SUMO is sufficient to silence the dimeric potassium channel K2P1.
Small ubiquitin modifier 1 (SUMO1) is shown to regulate K2P1 background channels in the plasma membrane (PM) of live mammalian cells. Confocal microscopy reveals native SUMO1, SAE1, and Ubc9 (the enzymes that activate and conjugate SUMO1) at PM where SUMO1 and expressed human K2P1 are demonstrated to colocalize. Silent K2P1 channels in excised PM patches are activated by SUMO isopeptidase (SENP1) and resilenced by SUMO1. K2P1-Lys274 is crucial: when mutated to Gln, Arg, Glu, Asp, Cys, or Ala, the channels are constitutively active and insensitive to SUMO1 and SENP1. Tandem mass spectrometry confirms conjugation of SUMO1 to the epsilon-amino group of Lys274 in vitro. FRET microscopy shows that assembly of K2P1 and SUMO1 requires Lys274. Single-particle TIRF microscopy shows that wild-type channels in PM have two K2P1 subunits and assemble with two SUMO1 monomers. Although channels engineered with one Lys274 site carry just one SUMO1 they are activated and silenced by SENP1 and SUMO1 like wild-type channels.
Plant
Leigh D
LD
Department of Pediatrics and Institute for Molecular Pediatric Sciences, Pritzker School of Medicine, Biological Sciences Division, University of Chicago, Chicago, IL 60637, USA.
Dementieva
Irina S
IS
Kollewe
Astrid
A
Olikara
Sonia
S
Marks
Jeremy D
JD
Goldstein
Steve A N
SA
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D024683:Potassium Channels, Tandem Pore Domain / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*
D024683:Potassium Channels, Tandem Pore Domain; D025842:SUMO-1 Protein