Proceedings of The Physiological Society
University of Edinburgh (2011) Proc Physiol Soc 25, PC38
Voltage-gated calcium channel antagonists block Nicotinic acid adenine dinucleotide phosphate-induced calcium signals via Two Pore Segment Channel subtype 2
O. A. Ogunbayo1, E. O. Agbani1, J. Ma2, M. X. Zhu3, M. A. Evans1
1. Centre For Integrative Physiology, University of Edinburgh, UK, Edinburgh, United Kingdom. 2. Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey, United States. 3. Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, Texas, United States.
Our recent proposal that nicotinic acid adenine dinucleotide phosphate (NAADP) mobilises acidic Ca2+ stores via two pore segment channels (TPCs, TPCN for gene name) has received significant support from subsequent studies (1, 2). TPCs represent evolutionary intermediates between the Voltage-Gated Ca2+ Channels and Transient Receptor Potential Channel families (3). Using methods described previously (1), we assessed the effect of two different voltage-gated Ca2+ channel antagonists on NAADP-dependent Ca2+ signalling via TPC2, namely a dihydropyridine, Nifedipine, and a phenylalkylamine, Verapamil. All procedures accorded with current UK legislation. When applied by intracellular dialysis from a patch-pipette (voltage-clamp mode, -40 mV holding potential), 10 nM NAADP evoked robust Ca2+ transients in both acutely isolated rat pulmonary arterial smooth muscle cells (PASMCs) and in HEK293 cells that stably over-expressed human (h) TPC2. The Fura-2 fluorescence ratio (F340/F380) increased from 0.65 ± 0.04 to 1.85 ± 0.14 (n=20) in acutely isolated rat PASMCs and from 0.31±0.02 to 1.11±0.06 (n=21) in hTPC2 expressing HEK293 cells, respectively. In both cell types, NAADP-evoked Ca2+ transients were markedly attenuated by thapsigargin (1μM), the F340/F380 ratio increasing from 0.55 ± 0.04 to 0.90 ± 0.03 (n=8) in PASMCs and from 0.25 ± 0.01 to 0.58 ± 0.03 (n=9) in hTPC2 expressing HEK293 cells, respectively. By contrast, evoked Ca2+ transients were abolished by bafilomycin (1μM), the F340/F380 increasing from 0.72 ± 0.04 to 0.87 ± 0.06 (n=7) in PASMCs and from 0.43 ± 0.05 to 0.45 ± 0.05 (n=3) in hTPC2 expressing HEK293 cells, respectively. Consistent with the effects of bafilomycin, but not thapsigargin, 10μM Nifedipine and 10μM Verapamil abolished Ca2+ transients in response to 10 nM NAADP in both cell types. In the presence of Nifedipine, the F340/F380 ratio increased from 0.35 ± 0.06 to 0.39 ± 0.06 (n=6) in PASMCs and from 0.43 ± 0.03 to 0.51 ± 0.04 (n=5) in hTPC2 expressing HEK293 cells, respectively. With Verapamil, F340/F380 increased from 0.32 ± 0.04 to 0.35 ± 0.06 (n=5) in PASMCs and from 0.4 ± 0.08 to 0.47 ± 0.08 (n=4) in hTPC2 expressing HEK293 cells, respectively. Using deconvolution microscopy we found that mcherry tagged hTPC2 was bound by extracellularly applied bodipy dihydrohypyridine (n=3). We conclude that both voltage-gated Ca2+ channel antagonists bind to and are effective blockers of hTPC2.
Where applicable, experiments conform with Society ethical requirements