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¹ Pacific Biosciences Research Center, University of Hawaii, Honolulu, HI 96822
² Laboratory of Cell and Molecular Signaling, Center for Biomedical Research at The Queen's Medical Center and John A. Burns School of Medicine at the University of Hawaii, Honolulu, HI 96813

Correspondence to Reinhold Penner: rpenner{at}hawaii.edu

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), Ca²⁺ 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 Ca²⁺ 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 Ca²⁺, both internally and externally, ADPR alone cannot induce cation currents. In the absence of extracellular Ca²⁺, a minimum of 30 nM internal Ca²⁺ is required to cause partial TRPM2 activation with ADPR. However, 200 µM external Ca²⁺ is as efficient as 1 mM Ca²⁺ in TRPM2 activation, indicating an external Ca²⁺ binding site important for proper channel function. Ca²⁺ facilitates ADPR gating with a half-maximal effective concentration of 50 nM and this is independent of extracellular Ca²⁺. Furthermore, TRPM2 currents inactivate if intracellular Ca²⁺ levels fall below 100 nM irrespective of extracellular Ca²⁺. The facilitatory effect of intracellular Ca²⁺ is not mimicked by Mg²⁺, Ba²⁺, or Zn²⁺. Only Sr²⁺ facilitates TRPM2 as effectively as Ca²⁺, but this is due to Sr²⁺-induced Ca²⁺ release from internal stores rather than a direct effect of Sr²⁺ itself. Together, these data demonstrate that cytosolic Ca²⁺ regulates TRPM2 channel activation. Its facilitatory action likely occurs via CaM, since the addition of 100 µM CaM to the patch pipette significantly enhances ADPR-induced TRPM2 currents at fixed [Ca²⁺]_i and this can be counteracted by calmidazolium. We conclude that ADPR is responsible for TRPM2 gating and Ca²⁺ facilitates activation via calmodulin.

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