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Dive into the research topics where Annette Lis is active.

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Featured researches published by Annette Lis.


Nature Cell Biology | 2006

Amplification of CRAC current by STIM1 and CRACM1 (Orai1)

Christine Peinelt; Monika Vig; Dana Lynn T Koomoa; Andreas Beck; Monica J. S. Nadler; Murielle Koblan-Huberson; Annette Lis; Andrea Fleig; Reinhold Penner; Jean-Pierre Kinet

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (ICRAC). However, individual overexpression of either protein fails to significantly amplify ICRAC. Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates ICRAC, suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.


Current Biology | 2006

CRACM1 Multimers Form the Ion-Selective Pore of the CRAC Channel

Monika Vig; Andreas Beck; James M. Billingsley; Annette Lis; Suhel Parvez; Christine Peinelt; Dana Lynn T Koomoa; Jonathan Soboloff; Donald L. Gill; Andrea Fleig; Jean-Pierre Kinet; Reinhold Penner

Receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) is often followed by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC) channels in the plasma membrane . RNAi screens have identified STIM1 as the putative ER Ca(2+) sensor and CRACM1 (Orai1; ) as the putative store-operated Ca(2+) channel. Overexpression of both proteins is required to reconstitute CRAC currents (I(CRAC); ). We show here that CRACM1 forms multimeric assemblies that bind STIM1 and that acidic residues in the transmembrane (TM) and extracellular domains of CRACM1 contribute to the ionic selectivity of the CRAC-channel pore. Replacement of the conserved glutamate in position 106 of the first TM domain of CRACM1 with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na(+), Ba(2+), and Sr(2+) permeation relative to Ca(2+). Mutating E190Q in TM3 also affects channel selectivity, suggesting that glutamate residues in both TM1 and TM3 face the lumen of the pore. Furthermore, mutating a putative Ca(2+) binding site in the first extracellular loop of CRACM1 (D110/112A) enhances monovalent cation permeation, suggesting that these residues too contribute to the coordination of Ca(2+) ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca(2+)-selective CRAC-channel pore.


Nature Cell Biology | 2008

Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells.

Thomas F. J. Wagner; Sabine Loch; Sachar Lambert; Isabelle Straub; Stefanie Mannebach; Ilka Mathar; Martina Düfer; Annette Lis; Veit Flockerzi; Stephan E. Philipp; Johannes Oberwinkler

Transient receptor potential (TRP) cation channels are renowned for their ability to sense diverse chemical stimuli. Still, for many members of this large and heterogeneous protein family it is unclear how their activity is regulated and whether they are influenced by endogenous substances. On the other hand, steroidal compounds are increasingly recognized to have rapid effects on membrane surface receptors that often have not been identified at the molecular level. We show here that TRPM3, a divalent-permeable cation channel, is rapidly and reversibly activated by extracellular pregnenolone sulphate, a neuroactive steroid. We show that pregnenolone sulphate activates endogenous TRPM3 channels in insulin-producing β cells. Application of pregnenolone sulphate led to a rapid calcium influx and enhanced insulin secretion from pancreatic islets. Our results establish that TRPM3 is an essential component of an ionotropic steroid receptor enabling unanticipated crosstalk between steroidal and insulin-signalling endocrine systems.


The Journal of Physiology | 2008

2‐Aminoethoxydiphenyl borate directly facilitates and indirectly inhibits STIM1‐dependent gating of CRAC channels

Christine Peinelt; Annette Lis; Andreas Beck; Andrea Fleig; Reinhold Penner

2‐Aminoethoxydiphenyl borate (2‐APB) has emerged as a useful pharmacological tool in the study of store‐operated Ca2+ entry (SOCE). It has been shown to potentiate store‐operated Ca2+ release‐activated Ca2+ (CRAC) currents at low micromolar concentrations and to inhibit them at higher concentrations. Initial experiments with the three CRAC channel subtypes CRACM1, CRACM2 and CRACM3 have indicated that they might be differentially affected by 2‐APB. We now present a thorough pharmacological profile of 2‐APB and report that it can activate CRACM3 channels in a store‐independent manner without the requirement of STIM1, whereas CRACM2 by itself is completely unresponsive to 2‐APB and CRACM1 is only very weakly activated. However, when coexpressed with STIM1 and activated via store depletion, CRACM1 and CRACM2 are facilitated at low 2‐APB concentrations and inhibited at higher concentrations, while CRACM3 only exhibits potentiated currents. Consistently, the 2‐APB‐induced CRAC currents exhibit altered selectivities that are characterized by a leftward shift in reversal potential and the emergence of large outward currents that are carried by normally impermeant monovalent cations such as Cs+ or K+. These results suggest that 2‐APB has agonistic and antagonistic modes of action on CRAC channels, acting at the channel level as a store‐independent and direct gating agonist for CRACM3 and a potentiating agonist for CRACM1 and CRACM2 following store‐operated and STIM1‐dependent activation. The inhibition of CRACM1 channels by high concentrations of 2‐APB appears to involve a direct block at the channel level and an additional uncoupling of STIM1 and CRACM1, since the compound reversed the store‐dependent multimerization of STIM1. Finally, we demonstrate that single‐point mutations of critical amino acids in the selectivity filter of the CRACM1 pore (E106D and E190A) enable 2‐APB to gate CRACM1 in a STIM1‐independent manner, suggesting that 2‐APB facilitates CRAC channels by altering the pore architecture.


The FASEB Journal | 2008

STIM2 protein mediates distinct store-dependent and store-independent modes of CRAC channel activation

Suhel Parvez; Andreas Beck; Christine Peinelt; Jonathan Soboloff; Annette Lis; Mahealani K. Monteilh-Zoller; Donald L. Gill; Andrea Fleig; Reinhold Penner

STIM1 and CRACM1 (or Orai1) are essential molecular components mediating store‐oper‐ated Ca2+ entry (SOCE) and Ca2+ release‐activated Ca2+ (CRAC) currents. Although STIM1 acts as a luminal Ca2+ sensor in the endoplasmic reticulum (ER), the function of STIM2 remains unclear. Here we reveal that STIM2 has two distinct modes of activating CRAC channels: a store‐operated mode that is activated through depletion of ER Ca2+ stores by inositol 1,4,5‐trisphosphate (InsP3) and store‐independent activation that is mediated by cell dialysis during whole‐cell perfusion. Both modes are regulated by calmodulin (CaM). The store‐operated mode is transient in intact cells, possibly reflecting recruitment of CaM, whereas loss of CaM in perfused cells accounts for the persistence of the store‐independent mode. The inhibition by CaM can be reversed by 2‐aminoethoxydiphenyl borate (2‐APB), resulting in rapid, store‐independent activation of CRAC channels. The aminoglycoside antibiotic G418 is a highly specific and potent inhibitor of STIM2‐dependent CRAC channel activation. The results reveal a novel bimodal control of CRAC channels by STIM2, the store dependence and CaM regulation, which indicates that the STIM2/CRACM1 complex may be under the control of both luminal and cytoplasmic Ca2+ levels.—Parvez S., Beck, A., Peinelt, C., Soboloff, J., Lis, A., Monteilh‐Zoller, M., Gill, D. L., Fleig, A., Penner R. STIM2 protein mediates distinct store‐dependent and store‐independent modes of CRAC channel activation. FASEB J. 22, 752–761 (2008)


The Journal of General Physiology | 2010

A single lysine in the N-terminal region of store-operated channels is critical for STIM1-mediated gating

Annette Lis; Susanna Zierler; Christine Peinelt; Andrea Fleig; Reinhold Penner

Store-operated Ca2+ entry is controlled by the interaction of stromal interaction molecules (STIMs) acting as endoplasmic reticulum ER Ca2+ sensors with calcium release–activated calcium (CRAC) channels (CRACM1/2/3 or Orai1/2/3) in the plasma membrane. Here, we report structural requirements of STIM1-mediated activation of CRACM1 and CRACM3 using truncations, point mutations, and CRACM1/CRACM3 chimeras. In accordance with previous studies, truncating the N-terminal region of CRACM1 or CRACM3 revealed a 20–amino acid stretch close to the plasma membrane important for channel gating. Exchanging the N-terminal region of CRACM3 with that of CRACM1 (CRACM3-N(M1)) results in accelerated kinetics and enhanced current amplitudes. Conversely, transplanting the N-terminal region of CRACM3 into CRACM1 (CRACM1-N(M3)) leads to severely reduced store-operated currents. Highly conserved amino acids (K85 in CRACM1 and K60 in CRACM3) in the N-terminal region close to the first transmembrane domain are crucial for STIM1-dependent gating of CRAC channels. Single-point mutations of this residue (K85E and K60E) eliminate store-operated currents induced by inositol 1,4,5-trisphosphate and reduce store-independent gating by 2-aminoethoxydiphenyl borate. However, short fragments of these mutant channels are still able to communicate with the CRAC-activating domain of STIM1. Collectively, these findings identify a single amino acid in the N terminus of CRAC channels as a critical element for store-operated gating of CRAC channels.


Journal of Immunology | 2005

Modulation of Ca2+ Signaling by Na+/Ca2+ Exchangers in Mast Cells

Eduardo Aneiros; Stephan E. Philipp; Annette Lis; Marc Freichel; Adolfo Cavalié

Mast cells rely on Ca2+ signaling to initiate activation programs leading to release of proinflammatory mediators. The interplay between Ca2+ release from internal stores and Ca2+ entry through store-operated Ca2+ channels has been extensively studied. Using rat basophilic leukemia (RBL) mast cells and murine bone marrow-derived mast cells, we examine the role of Na+/Ca2+ exchangers. Calcium imaging experiments and patch clamp current recordings revealed both K+-independent and K+-dependent components of Na+/Ca2+ exchange. Northern blot analysis indicated the predominant expression of the K+-dependent sodium-calcium exchanger NCKX3. Transcripts of the exchangers NCX3 and NCKX1 were additionally detected in RBL cells with RT-PCR. The Ca2+ clearance via Na+/Ca2+ exchange represented ∼50% of the total clearance when Ca2+ signals reached levels ≥200 nM. Ca2+ signaling and store-operated Ca2+ entry were strongly reduced by inverting the direction of Na+/Ca2+ exchange, indicating that Na+/Ca2+ exchangers normally extrude Ca2+ ions from cytosol and prevent the Ca2+-dependent inactivation of store-operated Ca2+ channels. Working in the Ca2+ efflux mode, Na+/Ca2+ exchangers such as NCKX3 and NCX3 might, therefore, play a role in the Ag-induced mast cell activation by controlling the sustained phase of Ca2+ mobilization.


Journal of Biological Chemistry | 2011

Transient receptor potential melastatin 1 (TRPM1) is an ion-conducting plasma membrane channel inhibited by zinc ions

Sachar Lambert; Anna Drews; Oleksandr Rizun; Thomas F. J. Wagner; Annette Lis; Stephanie Mannebach; Sandra Plant; Melanie Portz; Marcel Meissner; Stephan E. Philipp; Johannes Oberwinkler

TRPM1 is the founding member of the melastatin subgroup of transient receptor potential (TRP) proteins, but it has not yet been firmly established that TRPM1 proteins form ion channels. Consequently, the biophysical and pharmacological properties of these proteins are largely unknown. Here we show that heterologous expression of TRPM1 proteins induces ionic conductances that can be activated by extracellular steroid application. However the current amplitudes observed were too small to enable a reliable biophysical characterization. We overcame this limitation by modifying TRPM1 channels in several independent ways that increased the similarity to the closely related TRPM3 channels. The resulting constructs produced considerably larger currents after overexpression. We also demonstrate that unmodified TRPM1 and TRPM3 proteins form functional heteromultimeric channels. With these approaches, we measured the divalent permeability profile and found that channels containing the pore of TRPM1 are inhibited by extracellular zinc ions at physiological concentrations, in contrast to channels containing only the pore of TRPM3. Applying these findings to pancreatic β cells, we found that TRPM1 proteins do not play a major role in steroid-activated currents of these cells. The inhibition of TRPM1 by zinc ions is primarily due to a short stretch of seven amino acids present only in the pore region of TRPM1 but not of TRPM3. Combined, our data demonstrate that TRPM1 proteins are bona fide ion-conducting plasma membrane channels. Their distinct biophysical properties allow a reliable identification of endogenous TRPM1-mediated currents.


The Journal of Physiology | 2013

STIM2 drives Ca2+ oscillations through store-operated Ca2+ entry caused by mild store depletion

Markus Thiel; Annette Lis; Reinhold Penner

Stromal cell‐interaction molecule (STIM) 2 senses Ca2+ levels in the endoplasmic reticulum and activates Ca2+ channels in the plasma membrane upon store depletion. Here we report that STIM2 is preferentially activated by low agonist concentrations that cause mild reductions in endoplasmic reticulum Ca2+ levels. This shows that store‐operated Ca2+ entry is regulated through signal strength, with weak stimuli activating STIM2 and strong stimuli engaging STIM1. The results help us to understand how receptor activation enables differential modulation of Ca2+ entry over a range of agonist concentrations and levels of store depletion.


Journal of Biological Chemistry | 2012

Alternative Splicing of a Protein Domain Indispensable for Function of Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels

Julia Frühwald; Julia Camacho Londoño; Sandeep Dembla; Stefanie Mannebach; Annette Lis; Anna Drews; Ulrich Wissenbach; Johannes Oberwinkler; Stephan E. Philipp

Background: TRPM3 proteins form Ca2+ permeable ion channels involved in insulin secretion and pain perception. Results: A domain indispensable for TRPM3 channel function (ICF) is subject to alternative splicing. Conclusion: This domain contributes essentially to the formation of TRPM channels and removing it by splicing modulates TRPM3-mediated Ca2+ signaling. Significance: Alternative splicing of the ICF domain regulates biological functions attributed to TRPM3. TRPM3 channels form ionotropic steroid receptors in the plasma membrane of pancreatic β and dorsal root ganglion cells and link steroid hormone signaling to insulin release and pain perception, respectively. We identified and compared the function of a number of TRPM3 splice variants present in mouse, rat and human tissues. We found that variants lacking a region of 18 amino acid residues display neither Ca2+ entry nor ionic currents when expressed alone. Hence, splicing removes a region that is indispensable for channel function, which is called the ICF region. TRPM3 variants devoid of this region (TRPM3ΔICF), are ubiquitously present in different tissues and cell types where their transcripts constitute up to 15% of the TRPM3 isoforms. The ICF region is conserved throughout the TRPM family, and its presence in TRPM8 proteins is also necessary for function. Within the ICF region, 10 amino acid residues form a domain essential for the formation of operative TRPM3 channels. TRPM3ΔICF variants showed reduced interaction with other TRPM3 isoforms, and their occurrence at the cell membrane was diminished. Correspondingly, coexpression of ΔICF proteins with functional TRPM3 subunits not only reduced the number of channels but also impaired TRPM3-mediated Ca2+ entry. We conclude that TRPM3ΔICF variants are regulatory channel subunits fine-tuning TRPM3 channel activity.

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Reinhold Penner

The Queen's Medical Center

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Andrea Fleig

The Queen's Medical Center

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Andreas Beck

University of Hawaii at Manoa

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