An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca2+ signals - PubMed (original) (raw)

Activation of single Ca2+-permeable TPC2 channels by NAADP. A, typical recordings from excised inside-out patches from the plasma membrane of HEK cells expressing TPC2, TPC2ΔN, or TPC2AA and stimulated as indicated with NAADP (500 n

m

in the bathing solution). Cs+ was the charge carrier, and the holding potential was −60 mV; C denotes the closed state. Results are typical (from top to bottom) of 15, 17, 17, and 4 experiments, respectively. B, current (i)-voltage (V) relationship for the NAADP-activated channels in TPC2ΔN-expressing cells. Results are means ± S.E., n = 8. C, recording, typical of three experiments, from an excised inside-out patch from the plasma membrane of HEK cells expressing TPC2ΔN. NAADP (500 n

m

) was added and removed as indicated. C denotes the closed state. D, recording, typical of three similar records, from a patch excised from the plasma membrane of HEK cells expressing TPC2ΔN and treated with NAADP (500 n

m

) and _trans_-NED19 (100 n

m

) as indicated. Cs+ was the charge carrier, and the holding potential was −40 mV; C, O1, and O2 denote the closed state and openings of one and two channels, respectively. Note the presence of basal activity (_NP_o), which is increased ∼20-fold by the addition of NAADP; _trans_-NED19 inhibits both the basal and NAADP-evoked activity. In both C and D changes of media are accompanied by brief electrical spike artifacts. E, summary results showing _NP_o for excised patches of cells expressing TPC2ΔN or TPC2AA and stimulated with the indicated concentrations of NAADP (nanomolar) in the bathing solution. Results are means ± S.E., with n shown above each bar. F, record, typical of five similar records, from an excised patch expressing TPC2ΔN and with Ca2+ as the charge carrier. Pipette solution contained 500 n

m

NAADP, and the holding potential was −100 mV. C denotes the closed state. G, current-voltage relationship from records similar to those shown in F. Recordings were restricted to negative holding potentials to avoid activation of voltage-gated Ca2+ channels that may be endogenously expressed in HEK cells (34). Results are means ± S.E., n = 5.