AMPKα1-sensitivity of Orai1 and Ca(2+) entry in T - lymphocytes.

BACKGROUND/AIMS: T-lymphocyte activation and function critically depends on Ca(2+) signaling, which is regulated by store operated Ca(2+) entry (SOCE). Human and mouse T lymphocytes express AMP activated kinase AMPKα1, which is rapidly activated following elevation of cytosolic Ca(2+) concentration ([Ca(2+)]i) by treatment of the cells with Ca(2+) ionophore or following inhibition of endosomal Ca(2+) ATPase with thapsigargin. AMPK is further activated by triggering of the T cell antigen receptor (TCR). The present study explored whether AMPK influences Ca(2+) entry and Ca(2+)-sensitive regulation of T-lymphocyte function. METHODS: T-lymphocytes were isolated and cultured from AMPKα1-deficient (ampk(-/-)) mice and from their wildtype (ampk(+/+)) littermates. The phenotype of the cells was analysed by flow cytometry, [Ca(2+)]i estimated from Fura-2 fluorescence, SOCE from increase of [Ca(2+)]i following thapsigargin treatment (1 µM), and cell function analysed by measuring cytokine secretion and western blotting. RESULTS: Expression of surface markers in CD4(+) and CD8(+) T-cells were similar in ampk(-/-) and ampk(+/+) T-lymphocyte blasts. Moreover, total STIM1 protein abundance was similar in ampk(-/-) and ampk(+/+) T-lymphocyte blasts. However, Orai1 cell membrane protein abundance was significantly higher in ampk(-/-) than in ampk(+/+) T-lymphocyte blasts. SOCE and increase of [Ca(2+)]i following TCR activation by triggering TCR with anti-CD3 and cross-linking secondary antibody were both significantly more pronounced in ampk(-/-) than in ampk(+/+) T-lymphocyte blasts. The difference of Ca(2+) entry between ampk(-/-) and ampk(+/+) T-lymphocytes was abrogated by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-APB, 50 µM). Proliferation of unstimulated ampk(-/-) lymphocytes was higher than proliferation of ampk(+/+) T-lymphocytes, a difference reversed by Orai1 silencing. CONCLUSIONS: AMPK downregulates Orai1 and thus SOCE in T-lymphocytes and thus participates in negative feed-back regulation of cytosolic Ca(2+) activity.

Regulation of KCNQ1/KCNE1 by ß-catenin.

ß-catenin, a multifunctional protein expressed in all tissues including the heart stimulates the expression of several genes important for cell proliferation. Signaling involving ß-catenin participates in directing cardiac development and in the pathophysiology of cardiac hypertrophy. Nothing is known, however, on the role of ß-catenin in the regulation of cardiac ion channels. The present study explored the functional interaction of ß-catenin and KCNE1/KCNQ1, the K⁺ channel complex underlying the slowly activating outwardly rectifying K⁺ current. To this end, KCNE1/KCNQ1 was expressed in Xenopus oocytes with and without ß-catenin and the depolarization (up to + 80 mV) induced current (I(Ks)) was determined using the two-electrode voltage clamp. As a result, ß-catenin enhanced I(Ks) by 30%. The effect of ß-catenin on I(Ks) was not affected by actinomycin D (10 µM), an inhibitor of transcription, indicating that ß-catenin was not effective as transcription factor. Confocal microscopy revealed that ß-catenin enhanced the KCNE1/KCNQ1 protein abundance in the cell membrane. Exposure of the oocytes to brefeldin A (5 µM), an inhibitor of vesicle insertion, was followed by a decline of I(Ks), which was then similar in oocytes expressing KCNE1/KCNQ1 together with ß-catenin and in oocytes expressing KCNE1/KCNQ1 alone. In conclusion, ß-catenin enhances I(Ks) by increasing the KCNE1/KCNQ1 protein abundance in the cell membrane, an effect requiring vesicle insertion into the cell membrane.

Stimulation of Na+/K+ ATPase activity and Na+ coupled glucose transport by ß-catenin.

ß-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. ß-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that ß-catenin influences membrane transport. To this end, ß-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of ß-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na(+)/K(+)-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of ß-catenin on the endogenous Na(+)/K(+)-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of ß-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of ß-catenin expression. The stimulating effect of ß-catenin on both Na(+)/K(+) ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of ß-catenin, i.e. the regulation of transport.

Stimulation of HERG channel activity by ß-catenin.

The multifunctional protein ß-catenin governs as transcription factor the expression of a wide variety of genes relevant for cell proliferation and cell survival. In addition, ß-catenin is localized at the cell membrane and may influence the function of channels. The present study explored the possibility that ß-catenin participates in the regulation of the HERG K(+) channel. To this end, HERG was expressed in Xenopus oocytes with or without ß-catenin and the voltage-gated current determined utilizing the dual electrode voltage clamp. As a result, expression of ß-catenin markedly upregulated HERG channel activity, an effect not sensitive to inhibition of transcription with actinomycin D (10 µM). According to chemiluminescence, ß-catenin may increase HERG channel abundance within the oocyte cell membrane. Following inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) the decay of current was similar in oocytes expressing HERG together with ß-catenin to oocytes expressing HERG alone. The experiments uncover a novel function of APC/ß-catenin, i.e. the regulation of HERG channels.