Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases.

Although N-and P-type Ca 2؉ channels predominant in fast-secreting systems, Lc-type Ca 2؉ channels (C-class) can play a similar role in certain secretory cells and synapses. For example, in retinal bipolar cells, Ca 2؉ entry through the Lc channels triggers ultrafast exocytosis, and in pancreatic ␤-cells, evoked secretion is highly sensitive to Ca 2؉ . These findings suggest that a rapidly release pool of vesicles colocalizes with the Ca 2؉ channels to allow high Ca 2؉ concentration and a tight coupling of the Lc channels at the release site. In binding studies, we show that the Lc channel is physically associated with synaptotagmin (p65) and the soluble N-ethylmaleimide-sensitive attachment proteins receptors: syntaxin and synaptosomal-associated protein of 25 kDa. Soluble N-ethylmaleimide-sensitive attachent proteins receptors coexpressed in Xenopus oocytes along with the Lc channel modify the kinetic properties of the channel. The modulatory action of syntaxin can be overcome by coexpressing p65, where at a certain ratio of p65͞syntaxin, the channel regains its unaltered kinetic parameters. The cytosolic region of the channel, Lc 753-893 , separating repeats II-III of its ␣1C subunit, interacts with p65 and ''pulls'' down native p65 from rat brain membranes. Lc 753-893 injected into single insulinsecreting ␤-cell, inhibits secretion in response to channel opening, but not in response to photolysis of caged Ca 2؉ , nor does it affect Ca 2؉ current. These results suggest that Lc 753-893 competes with the endogenous channel for the synaptic proteins and disrupts the spatial coupling with the secretory apparatus. The molecular organization of the Lc channel and the secretory machinery into a multiprotein complex (named excitosome) appears to be essential for an effective depolarization evoked exocytosis.Regulated secretion in synapses occurs at a fast speed from vesicles preassembled with N-and P-type voltage sensitive Ca 2ϩ channels (1). In contrast, in many neuroendocrine cells exocytosis triggered by Ca 2ϩ entry through Lc channel, is slower and persists for tens of milliseconds after Ca 2ϩ influx has stopped, implying that the vesicles are localized at a distance from the source of Ca 2ϩ entry (2-4). Although exocytosis is slow in various endocrine cells in which secretion is mediated by Lc channel (C-class), there are reports suggesting a close association of Lc channels with the exocytotic machinery (5-8). For example a combined study of amperometry and laser imaging in chromaffin cells have shown that the sites of Ca 2ϩ entry and catecholamine release are close (5, 6). Similarly, in mouse pancreatic ␤-cells, Lc channels have been shown to colocalize with insulin-containing secretory granules (7). Previously, we showed that the expression of syntaxin, synaptosomal-associated protein of 25 kDa (SNAP-25), and p65 along with the L-and N-type channel modify the kinetic properties of the channels (8-10). The N-type Ca 2ϩ channel binds syntaxin and SNAP-25 (11-14) at a site in the cytoplasmic domain of ␣1...

We have used an electrophysiological assay to investigate the functional interaction of syntaxin 1A and SNAP‐25 with the class C, L‐type, and the class B, N‐type, voltage‐sensitive calcium channels. Co‐expression of syntaxin 1A with the pore‐forming subunits of the L‐ and N‐type channels in Xenopus oocytes generates a dramatic inhibition of inward currents (>60%) and modifies the rate of inactivation (tau) and steady‐state voltage dependence of inactivation. Syntaxin 1–267, which lacks the transmembrane region (TMR), and syntaxin 2 do not modify channel properties, suggesting that the syntaxin 1A interaction site resides predominantly in the TMR. Co‐expression of SNAP‐25 significantly modifies the gating properties of L‐ and N‐type channels and displays modest inhibition of current amplitude. Syntaxin 1A and SNAP‐25 combined restore the syntaxin‐inhibited N‐type inward current but not the reduced rate of inactivation. Hence, a distinct interaction of a putative syntaxin 1A‐SNAP‐25 complex with the channel is apparent, consistent with the formation of a synaptosomal SNAP receptors (SNAREs) complex. The in vivo functional reconstitution: (i) establishes the proximity of the SNAREs to calcium channels; (ii) provides new insight into a potential regulatory role for the two SNAREs in controlling calcium influx through N‐ and L‐type channels; and (iii) may suggest a pivotal role for calcium channels in the secretion process.

TASK3 gene (Kcnk9) is amplified and overexpressed in several types of human carcinomas. In this report, we demonstrate that a point mutation (G95E) within the consensus K ؉ filter of TASK3 not only abolished TASK3 potassium channel activity but also abrogated its oncogenic functions, including proliferation in low serum, resistance to apoptosis, and promotion of tumor growth. Furthermore, we provide evidence that TASK3 G95E is a dominant-negative mutation, because coexpression of the wild-type and the mutant TASK3 resulted in inhibition of K ؉ current of wild-type TASK3 and its tumorigenicity in nude mice. These results establish a direct link between the potassium channel activity of TASK3 and its oncogenic functions and imply that blockers for this potassium channel may have therapeutic potential for the treatment of cancers.T ASK (TWIK-related acid-sensitive K ϩ channels) channels are members of the two-pore domain family of potassium channels, whose structure consists of two-pore forming regions flanked by four transmembrane domains (1, 2). Like other two-pore domain members, these channels show little time or voltage dependence; thus, they have characteristics of leaky K ϩ channels, generating background currents that contribute to membrane potential and the regulation of cell excitability. The activity of TASK channels is modulated by volatile anesthetics (3, 4), neurotransmitters (5, 6), as well as extracellular pH in the physiological range (7-11). TASK3 is expressed at very low levels among the normal tissues except in the brain, where high levels expression of TASK3 were detected (7-9). The physiological functions of TASK channels are largely unknown, though their roles in the regulation of breathing (12, 13), aldosterone secretion (5) and anesthetic-mediated neuronal activity (14) have been proposed. Recently, we showed that TASK3 is amplified in 10% of breast cancers and is overexpressed at a higher frequency of breast, lung, colon, and metastatic prostate cancers (15), suggesting that TASK3 may play a role in pathogenesis of some human carcinomas.Is the dysregulated expression of TASK3 in tumor cells a consequence of their abnormal growth or is this K ϩ channel involved in promoting tumor growth? To begin to answer this question, we created an inactivating mutation of TASK3. We report here that TASK3 G95E is a dominant-negative mutation that abolishes not only TASK3 K ϩ channel activity but also its oncogenic functions. These results provide molecular basis for developing specific blockers for this K ϩ channel in the treatment of cancer. Materials and MethodsPlasmids and Mutagenesis. The coding region of TASK3 was cloned at BamHI and EcoRI sites of the pLPC retroviral expression vector (15) to generate pLPC-TASK3. Site-directed mutagenesis was performed to change Gly-95 to Glu to create pLPC-TASK3 G95E , by using QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. pTracer-TASK 3 G95E was generated by excising TASK3 G95E from pLPC vector and cloned at KpnI...

Excitatory synapses in the brain undergo activity-dependent changes in the strength of synaptic transmission. Such synaptic plasticity as exemplified by long-term potentiation (LTP) is considered a cellular correlate of learning and memory. The presence of G protein-activated inwardly rectifying K ؉ (GIRK) channels near excitatory synapses on dendritic spines suggests their possible involvement in synaptic plasticity. However, whether activitydependent regulation of GIRK channels affects excitatory synaptic plasticity is unknown. In a companion article we have reported activity-dependent regulation of GIRK channel density in cultured hippocampal neurons that requires activity of NMDA receptors (NMDAR) and protein phosphatase-1 (PP1) and takes place within 15 min. In this study, we performed whole-cell recordings of cultured hippocampal neurons and found that NMDAR activation increases basal GIRK current and GIRK channel activation mediated by adenosine A 1 receptors, but not GABAB receptors. Given the similar involvement of NMDARs, adenosine A1 receptors, and PP1 in depotentiation of LTP caused by low-frequency stimulation that immediately follows LTP-inducing high-frequency stimulation, we wondered whether NMDAR-induced increase in GIRK channel surface density and current may contribute to the molecular mechanisms underlying this specific depotentiation. Remarkably, GIRK2 null mutation or GIRK channel blockade abolishes depotentiation of LTP, demonstrating that GIRK channels are critical for depotentiation, one form of excitatory synaptic plasticity.adenosine receptor ͉ synaptic plasticity ͉ learning and memory ͉ protein phosphatase-1 ͉ extracellular field recording S ynaptic plasticity, the ability of neurons to modify the efficacy of synaptic transmission, is thought to provide the cellular basis for the profound influence of experience over information processing and storage in the brain (1, 2). For example, long-term potentiation (LTP), a long-lasting increase in excitatory synaptic strength after heightened synaptic activity (3), is believed to be the cellular correlate of learning and memory. Since the discovery of LTP in the hippocampus, a region known to be essential for learning and memory, LTP has been extensively characterized for the molecular mechanisms of its induction and expression that involves Ca 2ϩ influx through NMDA receptors (NMDARs), leading to a high level of intracellular Ca 2ϩ concentration, and activation of calcium/calmodulin-dependent kinase II (CaMKII) (3).Whether the excitatory synaptic activities generated by highfrequency stimulation (HFS) result in LTP depends on the pattern of synaptic inputs impinging on the postsynaptic CA1 neurons shortly afterward; LTP of field excitatory postsynaptic potential (fEPSP) of CA1 neurons fails to develop if the HFS of the Schaffer collateral nerve fibers is followed within minutes by low-frequency stimulation (LFS) (4, 5). This form of synaptic plasticity, called depotentiation, may be a mechanism to abort the LTP according to events that take...

No abstract

G protein-activated inwardly rectifying potassium (GIRK) channels mediate slow synaptic inhibition and control neuronal excitability. It is unknown whether GIRK channels are subject to regulation by guanine dissociation inhibitor (GDI) proteins like LGN, a mammalian homolog of Drosophila Partner of Inscuteable (mPINS). Here we report that LGN increases basal GIRK current but reduces GIRK activation by metabotropic transmitter receptors coupled to Gi or Go, but not Gs. Moreover, expression of its N-terminal, TPR-containing protein interaction domains mimics the effects of LGN in mammalian cells, probably by releasing sequestered endogenous LGN. In hippocampal neurons, expression of LGN, or LGN fragments that mimic or enhance LGN activity, hyperpolarizes the resting potential due to increased basal GIRK activity and reduces excitability. Using Lenti virus for LGN RNAi to reduce endogenous LGN levels in hippocampal neurons, we further show an essential role of LGN for maintaining basal GIRK channel activity and for harnessing neuronal excitability.

Syntaxin 1A (Sx1A) modifies the activity of voltagegated Ca 2؉ channels acting via the cytosolic and the two vicinal cysteines (271 and 272) at the transmembrane domain. Here we show that Sx1A modulates the Lc-type Ca 2؉ channel, Ca v 1.2, in a cooperative manner, and we explore whether channel clustering or the Sx1A homodimer is responsible for this activity. Sx1A formed homodimers but, when mutated at the two vicinal transmembrane domain cysteines, was unable to either dimerize or modify the channel activity suggesting disulfide bond formation. Moreover, applying global molecular dynamic search established a theoretical prospect of generating a disulfide bond between two Sx1A transmembrane helices. Nevertheless, Sx1A activity was not correlated with Sx1A homodimer. Application of a vicinal thiol reagent, phenylarsine oxide, abolished Sx1A action indicating the accessibility of Cys-271,272 thiols. Sx1A inhibition of channel activity was restored by phenylarsine oxide antidote, 2,3-dimercaptopropanol, consistent with thiol interaction of Sx1A. In addition, the supralinear mode of channel inhibition was correlated to the monomeric form of Sx1A and was apparent only when the three channel subunits ␣ 1 1.2/␣ 2 ␦1/ ␤2a were present. This functional demonstration of cooperativity suggests that the three-subunit channel responds as a cluster, and Sx1A monomers associate with a dimer (or more) of a three-subunit Ca 2؉ channel. Consistent with channel cluster linked to Sx1A, a conformational change driven by membrane depolarization and Ca 2؉ entry would rapidly be transduced to the exocytotic machinery. As shown herein, the supralinear relationship between Sx1A and the voltage-gated Ca 2؉ channel within the cluster could convey the cooperativity that distinguishes the process of neurotransmitter release.Signal transduction in the synapse is initiated by neurotransmitter release as a consequence of fusion that takes place between synaptic vesicles and the plasma membrane. It is commonly accepted that at the heart of this tightly regulated, multifarious process lies the ternary complex formed by the synaptic proteins syntaxin 1A (Sx1A), 1 SNAP-25, and synaptobrevin II (1-3). This ternary complex is thought to be responsible for juxtaposing the synaptic vesicle and the plasma membrane prior to membrane fusion, which involves the binding of soluble N-ethylmaleimide-sensitive factor (NSF) (1-3). It is for this reason that the above-mentioned ternary complex is referred to as the SNARE (receptor for soluble NSF attachment proteins) complex.Analysis of ternary complexes formed by the full-length proteins revealed that the C-terminal transmembrane domains (TMDs) of both Sx1A and synaptobrevin II were protected from trypsin digestion (4). Moreover, inclusion of these TMDs increased the stability of the ternary complexes and affected the ability of the cytoplasmic domains to join other proteins (4, 5). Co-reconstituted into liposomes, synaptobrevin II and syntaxin 1A formed a stable binary complex that required the presence of th...