Dataset Information

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy.

ABSTRACT: Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca(2+) concentration inside pancreatic acinar cells ([Ca(2+)]i), due to excessive release of Ca(2+) stored inside the cells followed by Ca(2+) entry from the interstitial fluid. The sustained [Ca(2+)]i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca(2+) release-activated Ca(2+) channels (CRAC) would prevent sustained elevation of [Ca(2+)]i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca(2+) ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca(2+). Ca(2+) entry then occurred upon admission of Ca(2+) to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca(2+) entry in a concentration-dependent manner within the range of 1 to 50 ?M (IC50 = 3.4 ?M), but had little or no effect on the physiological Ca(2+) spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 ?M), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca(2+)]i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca(2+)]i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.

SUBMITTER: Gerasimenko JV

PROVIDER: S-EPMC3740877 | biostudies-other | 2013 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy.

Gerasimenko Julia V JV Gryshchenko Oleksiy O Ferdek Pawel E PE Stapleton Eloise E Hébert Tania O G TO Bychkova Solomiia S Peng Shuang S Begg Malcolm M Gerasimenko Oleg V OV Petersen Ole H OH

Proceedings of the National Academy of Sciences of the United States of America 20130722 32

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca(2+) concentration inside pancreatic acinar cells ([Ca(2+)]i), due to excessive release of Ca(2+) stored inside the cells followed by Ca(2+) entry from the interstitial fluid. The sustained [Ca(2+)]i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pha ...[more]

PMID: 23878235

Similar Datasets

Project description:Orai1, the pore subunit of Ca(2+) release-activated Ca(2+) channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca(2+) with a high affinity similar to the wild type, but the Ca(2+)-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 μm Ca(2+), more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca(2+), W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca(2+). These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca(2+) inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.

Project description:Prevailing models postulate that high Ca(2+) selectivity of Ca(2+) release-activated Ca(2+) (CRAC) channels arises from tight Ca(2+) binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca(2+) binding for Ca(2+) selectivity in recombinant Orai3 channels, which function as highly Ca(2+)-selective channels when gated by the endoplasmic reticulum Ca(2+) sensor STIM1 or as poorly Ca(2+)-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca(2+) blocked Na(+) currents in both gating modes with a similar inhibition constant (Ki; ~25 µM). Thus, equilibrium binding as set by the Ki of Ca(2+) blockade cannot explain the differing Ca(2+) selectivity of the two gating modes. Unlike STIM1-gated channels, Ca(2+) blockade in 2-APB-gated channels depended on the extracellular Na(+) concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na(+) pore occupancy. Moreover, the second-order rate constants of Ca(2+) blockade were eightfold faster in 2-APB-gated channels than in STIM1-gated channels. A four-barrier, three-binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca(2+) and Na(+) to simulate the faster rate constants of 2-APB-gated channels qualitatively reproduces their low Ca(2+) selectivity, suggesting that ion entry and exit rates strongly affect Ca(2+) selectivity. Noise analysis indicated that the unitary Na(+) conductance of 2-APB-gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (Po; ~0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high Po state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca(2+) binding and kinetic factors contribute to high Ca(2+) selectivity in CRAC channels.

Dataset Information

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy.

Publications

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets