Regulation of TRPM2 by extra- and intracellular calcium.
TRPM2 is a calcium-permeable nonselective cation channel that is opened by the binding of ADP-ribose (ADPR) to a C-terminal nudix domain. Channel activity is further regulated by several cytosolic factors, including cyclic ADPR (cADPR), nicotinamide adenine dinucleotide phosphate (NAADP), Ca(2+) and calmodulin (CaM), and adenosine monophosphate (AMP). In addition, intracellular ions typically used in patch-clamp experiments such as Cs(+) or Na(+) can alter ADPR sensitivity and voltage dependence, complicating the evaluation of the roles of the various modulators in a physiological context. We investigated the roles of extra- and intracellular Ca(2+) as well as CaM as modulators of ADPR-induced TRPM2 currents under more physiological conditions, using K(+)-based internal saline in patch-clamp experiments performed on human TRPM2 expressed in HEK293 cells. Our results show that in the absence of Ca(2+), both internally and externally, ADPR alone cannot induce cation currents. In the absence of extracellular Ca(2+), a minimum of 30 nM internal Ca(2+) is required to cause partial TRPM2 activation with ADPR. However, 200 microM external Ca(2+) is as efficient as 1 mM Ca(2+) in TRPM2 activation, indicating an external Ca(2+) binding site important for proper channel function. Ca(2+) facilitates ADPR gating with a half-maximal effective concentration of 50 nM and this is independent of extracellular Ca(2+). Furthermore, TRPM2 currents inactivate if intracellular Ca(2+) levels fall below 100 nM irrespective of extracellular Ca(2+). The facilitatory effect of intracellular Ca(2+) is not mimicked by Mg(2+), Ba(2+), or Zn(2+). Only Sr(2+) facilitates TRPM2 as effectively as Ca(2+), but this is due to Sr(2+)-induced Ca(2+) release from internal stores rather than a direct effect of Sr(2+) itself. Together, these data demonstrate that cytosolic Ca(2+) regulates TRPM2 channel activation. Its facilitatory action likely occurs via CaM, since the addition of 100 microM CaM to the patch pipette significantly enhances ADPR-induced TRPM2 currents at fixed [Ca(2+)](i) and this can be counteracted by calmidazolium. We conclude that ADPR is responsible for TRPM2 gating and Ca(2+) facilitates activation via calmodulin.
Starkus
John
J
Pacific Biosciences Research Center, University of Hawaii, Honolulu, HI 96822, USA.
Beck
Andreas
A
Fleig
Andrea
A
Penner
Reinhold
R
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling; D002147:Calmodulin; D002413:Cations, Divalent / Q000378:metabolism; D002461:Cell Line, Transformed; D036563:Cyclic ADP-Ribose / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D025461:Feedback, Physiological / Q000187:drug effects; D006801:Humans; D015640:Ion Channel Gating; D017136:Ion Transport; D018408:Patch-Clamp Techniques; D050053:TRPM Cation Channels / Q000378:metabolism*
D002120:Calcium Channel Agonists; D002147:Calmodulin; D002413:Cations, Divalent; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D036563:Cyclic ADP-Ribose; D002118:Calcium