In pancreatic ␤-cells, the predominant voltage-gated Ca 2؉ channel (Ca V 1.2) and K ؉ channel (K V 2.1) are directly coupled to SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor) proteins. These SNARE proteins modulate channel expression and gating and closely associate these channels with the insulin secretory vesicles. We show that K V 2.1 and Ca V 1.2, but not K V 1.4, SUR1, or Kir6.2, target to specialized cholesterol-rich lipid raft domains on ␤-cell plasma membranes. Similarly, the SNARE proteins syntaxin 1A, SNAP-25, and VAMP-2, but not Munc-13-1 or n-Sec1, are associated with lipid rafts. Disruption of the lipid rafts by depleting membrane cholesterol with methyl-␤-cyclodextrin shunts K V 2.1, Ca V 1.2, and SNARE proteins out of lipid rafts. Furthermore, methyl-␤-cyclodextrin inhibits K V 2.1 but not Ca V 1.2 channel activity and enhances single-cell exocytic events and insulin secretion. Membrane compartmentalization of ion channels and SNARE proteins in lipid rafts may be critical for the temporal and spatial coordination of insulin release, forming what has been described as the excitosome complex.In the pancreatic islets of Langerhans, glucose uptake by ␤-cells initiates a cascade of cellular events resulting in insulin secretion. A key response leading to insulin release is the change in transmembrane potential associated with the opening and closing of ion channels. Glucose uptake and metabolism increases the ratio of ATP/ADP, leading to the blockade of ATP-sensitive potassium (K ϩ -ATP) channels. Inhibition of these channels results in cell membrane depolarization and subsequent activation of voltage-gated Ca 2ϩ (Ca V ) 1 channels. Influx of extracellular Ca 2ϩ through Ca V channels causes oscillatory elevations in [Ca 2ϩ ] i , fusion of insulin-containing vesicles with the cell membrane, and insulin release (reviewed in Ref. 1). This entire process is suppressed or terminated by the opening of voltage-gated K ϩ (K V ) channels (2). The integrated process of channel gating is critical for the coordination of insulin release and thus the consequent maintenance of proper plasma glucose levels.Pancreatic ␤-cells and clonal insulinoma cells express four different families of K V channels (K V 1, K V 2, K V 3, K V 4) in variable levels (2-4). K V 2.1 is the most abundant K V channel isoform expressed in both isolated islet ␤-cells and insulinoma cells. To support this notion, the dominant-negative knockout of K V 2.1 channel or pharmacological blockade with a selective K V 2.1 antagonist reduces steady-state outward K V currents by ϳ60 -70% (2, 5). In addition to K V 2.1, other K V channel ␣ subunits are expressed in pancreatic ␤-cells to a lesser extent, including K V 1.4 and K V 1.6, which account for less than 25% of outward K ϩ currents measured in these cells (2). The central role of Ca V channels in insulin secretion is well recognized (1). The predominant Ca V channel in ␤-cells is the L-type channel (long-lasting; Ca V 1.2/␣ 1C-a and Ca V 1.3/␣ 1D ) (6, 7). T...

Acquisition of additional genetic and/or epigenetic abnormalities other than the BCR/ABL fusion gene is believed to cause disease progression in chronic myeloid leukemia (CML) from chronic phase to blast crisis (BC). To gain insights into the underlying mechanisms of progression to BC, we screened DNA samples from CML patients during blast transformation for mutations in a number of transcription factor genes that are critical for myeloid-lymphoid development. In 85 cases of CML blast transformation, we identified two new mutations in the coding region of GATA-2, a negative regulator of hematopoietic stem/progenitor cell differentiation. A L359V substitution within zinc finger domain (ZF) 2 of GATA-2 was found in eight cases with myelomonoblastic features, whereas an in-frame deletion of 6 aa (⌬341-346) spanning the C-terminal border of ZF1 was detected in one patient at myeloid BC with eosinophilia. Further studies indicated that L359V not only increased transactivation activity of GATA-2 but also enhanced its inhibitory effects on the activity of PU.1, a major regulator of myelopoiesis. Consistent with the myelomonoblastic features of CML transformation with the GATA-2 L359V mutant, transduction of the GATA-2 L359V mutant into HL-60 cells or BCR/ABL-harboring murine cells disturbed myelomonocytic differentiation/proliferation in vitro and in vivo, respectively. These data strongly suggest that GATA-2 mutations may play a role in acute myeloid transformation in a subset of CML patients.blast crisis ͉ chronic phase ͉ genetic alteration ͉ transcriptional regulation

Voltage-gated K؉ (Kv) 2.1 is the dominant Kv channel that controls membrane repolarization in rat islet ␤-cells and downstream insulin exocytosis. We recently showed that exocytotic SNARE protein SNAP-25 directly binds and modulates rat islet ␤-cell Kv 2.1 channel protein at the cytoplasmic N terminus. We now show that SNARE protein syntaxin 1A (Syn-1A) binds and modulates rat islet ␤-cell Kv2.1 at its cytoplasmic C terminus (Kv2.1C). In HEK293 cells overexpressing Kv2.1, we observed identical effects of channel inhibition by dialyzed GST-Syn-1A, which could be blocked by Kv2.1C domain proteins (C1: amino acids 412-633, C2: amino acids 634 -853), but not the Kv2.1 cytoplasmic N terminus (amino acids 1-182). This was confirmed by direct binding of GST-Syn-1A to the Kv2.1C1 and C2 domains proteins. These findings are in contrast to our recent report showing that Syn-1A binds and modulates the cytoplasmic N terminus of neuronal Kv1.1 and not by its C terminus. Co-expression of Syn-1A in Kv2.1-expressing HEK293 cells inhibited Kv2.1 surfacing, which caused a reduction of Kv2.1 current density. In addition, Syn-1A caused a slowing of Kv2.1 current activation and reduction in the slope factor of steady-state inactivation, but had no affect on inactivation kinetics or voltage dependence of activation. Taken together, SNAP-25 and Syn-1A mediate secretion not only through its participation in the exocytotic SNARE complex, but also by regulating membrane potential and calcium entry through their interaction with Kv and Ca 2؉ channels. In contrast to Ca 2؉ channels, where these SNARE proteins act on a common synprint site, the SNARE proteins act not only on distinct sites within a Kv channel, but also on distinct sites between different Kv channel families.

Purpose: NOTCH signaling pathway is essential in T-cell development and NOTCH1 mutations are frequently present inT-cell acute lymphoblastic leukemia (T-ALL). To gain insight into its clinical significance, NOTCH1 mutation was investigated in 77 patients withT-ALL. Experimental Design: Detection of NOTCH1 mutation was done using reverse transcription-PCR amplification and direct sequencing, and thereby compared according to the clinical/ biological data of the patients. Results: Thirty-two mutations were identified in 29 patients (with dual mutations in 3 cases), involving not only the heterodimerization and proline/glutamic acid/serine/threonine domains as previously reported but also the transcription activation and ankyrin repeat domains revealed for the first time. These mutations were significantly associated with elevated WBC count at diagnosis and independently linked to short survival time. Interestingly, the statistically significant difference of survival according to NOTCH1 mutations was only observed in adult patients (>18 years) but not in pediatric patients (V18 years), possibly due to the relatively good overall response of childhoodT-ALL to the current chemotherapy. NOTCH1 mutations could coexist with HOX11, HOX11L2, or SIL-TAL1 expression. The negative effect of NOTCH1 mutation on prognosis was potentiated by HOX11L2 but was attenuated by HOX11. Conclusion: NOTCH1 mutation is an important prognostic marker in T-ALL and its predictive value could be even further increased if coevaluated with other T-cell-related regulatory genes. NOTCH pathway thus acts combinatorially with oncogenic transcriptional factors on T-ALL pathogenesis.