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), 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.