Franklin M. Mullins

STIM1 Clusters and Activates CRAC Channels via Direct Binding of a Cytosolic Domain to Orai1

Store-operated Ca(2+) channels activated by the depletion of Ca(2+) from the endoplasmic reticulum (ER) are a major Ca(2+) entry pathway in nonexcitable cells and are essential for T cell activation and adaptive immunity. After store depletion, the ER Ca(2+) sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here, we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N and C termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca(2+) entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions.

STIM1 and calmodulin interact with Orai1 to induce Ca2+-dependent inactivation of CRAC channels

Ca2+-dependent inactivation (CDI) is a key regulator and hallmark of the Ca2+ release-activated Ca2+ (CRAC) channel, a prototypic store-operated Ca2+ channel. Although the roles of the endoplasmic reticulum Ca2+ sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342–448) that binds directly to Orai1 to activate the channel. Surprisingly, CAD-induced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470–491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68–91) that binds CaM in a Ca2+-dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.

Identification of an intronic single nucleotide polymorphism leading to allele dropout during validation of a CDH1 sequencing assay: implications for designing polymerase chain reaction-based assays

Purpose: The CDH1 gene encodes the cell adhesion protein E-cadherin, and CDH1 germline mutations are associated with hereditary diffuse gastric cancer. Identification of individuals at high risk of developing diffuse gastric cancer affords the opportunity for endoscopic screening or elective prophylactic gastrectomy. We set out to develop a CDH1 sequencing assay for clinical use.Methods: All exons of the CDH1 gene were amplified and sequenced with published and modified primers.Results: While validating the assay, we encountered a case in which a single nucleotide polymorphism located in intron 15 led to allele dropout and therefore to a false-negative result. The polymorphism leading to allele dropout was located within a primer-binding sequence, five bases away from the 3′ end of the primer. A frameshift mutation in exon 15 was detected by an alternative primer that binds away from the polymorphic site. A search of the University of California Santa Cruz single nucleotide polymorphism database revealed other polymorphisms located within primer-binding sites. A total of 12 primers in nine primer sets were modified to minimize allele dropout risk.Conclusion: The approach of designing primers to avoid known single nucleotide polymorphisms can be generalized to the design of any polymerase chain reaction-based assay and should be employed whenever possible.

Orai1 pore residues control CRAC channel inactivation independently of calmodulin.

Researchers have reevaluated the role of calmodulin and previously identified calmodulin binding sites in the mechanism by which Ca2+-release activated Ca2+ channels can be inactivated as Ca2+ ions enter cells.

The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca2+-dependent inactivation

Researchers examined the role of a domain within the stromal interaction molecule (STIM) in the mechanism by which Ca2+ release–activated Ca2+ channels can be inactivated as Ca2+ ions enter cells.

Cytologic evaluation of lymphadenopathy associated with mycosis fungoides and Sezary syndrome: role of immunophenotypic and molecular ancillary studies.

The most common presenting site of extracutaneous disease in mycosis fungoides and Sezary syndrome is the peripheral lymph node. Although fine‐needle aspiration biopsy has been shown to be a valuable diagnostic technique in evaluating lymphadenopathy, its utility in patients with cutaneous T‐cell lymphoma has not been extensively studied. With fine‐needle aspiration biopsy, material can be collected for ancillary diagnostic studies and for morphologic evaluation.

Hereditary diffuse gastric cancer due to a previously undescribed CDH1 splice site mutation

Our patient was a 52-year-old man who was diagnosed with signet ring cell gastric adenocarcinoma. An extensive family history of gastric cancer raised suspicion for hereditary diffuse gastric cancer. Sequencing of the patients CDH1 gene revealed a novel point mutation in a strictly conserved splice site within intron 6, c.833-2 A > G. This mutation was predicted to result in loss of function due to defective RNA splicing. To characterize the pathogenic mechanism of this mutation, we amplified the patients CDH1 gene products by reverse transcriptase polymerase chain reaction. Primers flanking the region of the mutation detected 3 distinct transcripts. In addition to the wild-type product, a larger product consistent with activation of a cryptic splice site within intron 6 and a smaller product shown to result from exon 7 skipping were detected. In summary, we have identified a novel CDH1 mutation in a large hereditary diffuse gastric cancer kindred and identified its pathogenic mechanism.