Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca2+ signalling and many other cellular processes. A calcium-release-activated calcium (CRAC) channel in T lymphocytes is the best-characterized SOC influx channel and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes. Here we show that the expression of EF-hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca2+ store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulum-like sites to the plasma membrane upon depletion of the Ca2+ store. We propose that STIM1 functions as the missing link between Ca2+ store depletion and SOC influx, serving as a Ca2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.

Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca 2؉ influx and Ca 2؉ release-activated Ca 2؉ (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca 2؉ influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca 2؉ entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca 2؉ pump sarco-͞ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca 2؉ depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.capacitative calcium entry (CCE) ͉ genome-wide screen ͉ CRAC channel ͉ RNA interference ͉ store-operated calcium (SOC) influx P atch-clamp experiments have identified the biophysical characteristics of Ca 2ϩ release-activated Ca 2ϩ (CRAC) channels in lymphocytes and other human cell types (1, 2). Despite the acknowledged functional importance of storeoperated Ca 2ϩ (SOC) influx in cell biology (2) and of CRAC channels for immune cell activation (3), the intrinsic channel components and signaling pathways that lead to channel activation remain unidentified. In previous work (4), we demonstrated that SOC influx in S2 cells occurs through a channel that shares biophysical properties with CRAC channels in human T lymphocytes. In a medium-throughput RNA interference (RNAi) screen targeting 170 candidate genes in S2 cells, we discovered an essential conserved role of Stim and the mammalian homolog STIM1 in SOC influx and CRAC channel activity (5). STIM1 and STIM2 also were identified in an independently performed screen of HeLa cells by using the Drosophila enzyme Dicer to generate small interfering RNA species from dsRNA (6). Drosophila Stim and the mammalian homolog STIM1 appear to play dual roles in the CRAC channel activation sequence, sensing the luminal Ca 2ϩ store content through an EF hand motif and trafficking from an endoplasmic reticulum (ER)-like localization to the plasma membrane to trigger CRAC channel activity (6-8). However, as single-pass transmembrane proteins, Stim and its mammalian homolog STIM1 are unlikely to form the CRAC channel itself. To search systematically for additional components of the CRAC channel, and to analyze the signaling network and other required factors th...

Peripheral nerve injury can lead to a persistent neuropathic pain state in which innocuous tactile stimulation elicits pain behavior (tactile allodynia). Spinal administration of the anticonvulsant gabapentin suppresses allodynia by an unknown mechanism. In vitro studies indicate that gabapentin binds to the ␣ 2 ␦-1 (hereafter referred to as ␣ 2 ␦) subunit of voltage-gated calcium channels. We hypothesized that nerve injury may result in altered ␣ 2 ␦ subunit expression in spinal cord and dorsal root ganglia (DRGs) and that this change may play a role in neuropathic pain processing. Using a rat neuropathic pain model in which gabapentin-sensitive tactile allodynia develops after tight ligation of the left fifth and sixth lumbar spinal nerves, we found a Ͼ17-fold, time-dependent increase in ␣ 2 ␦ subunit expression in DRGs ipsilateral to the nerve injury. Marked ␣ 2 ␦ subunit upregulation was also evident in rats with unilateral sciatic nerve crush, but not dorsal rhizotomy, indicating a peripheral origin of the expression regulation. The increased ␣ 2 ␦ subunit expression preceded the allodynia onset and diminished in rats recovering from tactile allodynia. RNase protection experiments indicated that the DRG ␣ 2 ␦ regulation was at the mRNA level. In contrast, calcium channel ␣ 1B and ␤ 3 subunit expression was not co-upregulated with the ␣ 2 ␦ subunit after nerve injury. These data suggest that DRG ␣ 2 ␦ regulation may play an unique role in neuroplasticity after peripheral nerve injury that may contribute to allodynia development. Key words: ␣ 2 ␦ calcium channel subunit; peripheral nerve injury; rhizotomy; allodynia; dorsal root ganglia; spinal cord; sensory neuronsPeripheral nerve injury may lead to neuropathic syndromes characterized by both spontaneous and evoked painful sensations. Allodynia, or an exaggerated response to otherwise innocuous tactile stimuli, is considered both a hallmark and the most troublesome of these syndromes (Price et al., 1989;Wahren and Torebjork, 1992;Koltzenburg et al., 1994). The molecular mechanisms of neuropathic pain states are not clear. It has been hypothesized that disordered sensory processing arises from long-term changes in the function of sensory afferents and the organization of sensory input into the dorsal horn (Coderre et al., 1993;Woolf and Doubell, 1994).Pharmacological evidence suggests that spinal N-type voltagegated calcium channels (N-VGCCs) play a role in neuropathic pain transduction. Intrathecal delivery of N-type ( -conopeptides), but not L-or P-type, VGCC antagonists suppresses allodynia in neuropathic rat models (Chaplan et al., 1994b;Calcutt and Chaplan, 1997). Autoradiographic studies showed the highest density of conopeptide-binding sites in the spinal dorsal horn (lamina I and II) where primary afferents terminate (Kerr et al., 1988;Takemura et al., 1989). N-VGCCs are also found in dorsal root ganglion (DRG) neurons where they are differentially modulated after sciatic nerve damage (Abdulla and Smith, 1997).High-threshold neuronal VGCCs contain thr...

Summary Alzheimer's disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta42 levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta40 levels while concomitantly elevating levels of Abeta38 and Abeta37. Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta42; chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g. intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).