phosphoinositide ͉ PI(4,5)P2 ͉ voltage sensor ͉ PI(3,4,5)P3 ͉ substrate specificity P hosphatidylinositol (PI) lipids serve structural roles in biological membranes as well as playing important roles as signaling molecules. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] regulates cell motility, cell shape, vesicle turnover, and membrane excitability either through directly binding to target proteins (1-3) or by mediating calcium signaling via its cleavage by phospholipase C into inositol 1,4,5-trisphosphate and diacylglycerol (4). Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] regulates cell proliferation, survival, and morphology (5). To exert physiological roles, the concentrations of these phosphoinositides in membranes are strictly regulated by multiple kinases and phosphatases. Recently, we identified a phosphoinositide phosphatase, Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP), consisting of an ion channel-like transmembrane domain followed by a phosphatase domain which shares sequence identity to the phosphatase and tensin homolog deleted on chromosome 10q23 (PTEN). PTEN is a well characterized PI phosphatase that dephosphorylates the phosphate on the 3Ј position of PI(3,4,5)P 3 , resulting in generation of PI(4,5)P 2 (6, 7). Ci-VSP shares overlapping substrate specificity with PTEN in that it was also shown to dephosphorylate PI(3,4,5)P 3 (8).A voltage-gated ion channel commonly consists of two parts: the N terminus (S1 to S4) functions as a voltage sensor while the C terminus (S5 to S6) functions as an ion-permeable pore (9). The transmembrane region of Ci-VSP shows significant sequence homology to the voltage-sensor domains of the conventional voltage-gated channels but does not contain the pore domain. Basic amino acids spaced periodically in the fourth transmembrane segments (S4) are known to be essential for sensing changes in the membrane potential in voltage-gated ion channels (8,9). This pattern of basic amino acids is also conserved in the fourth transmembrane segment (S4) of Ci-VSP. Indeed, Xenopus oocytes injected with Ci-VSP cRNA showed transient ''gating'' currents as the readouts of the movement of S4 across the membrane in response to voltage change, demonstrating that the N terminus of Ci-VSP functions as a voltage sensor (8).We hypothesized that the voltage-sensor domain of Ci-VSP could potentially regulate the activity of the phosphatase domain. Along these lines we were able to demonstrate that the activity of the PI(4,5)P 2 -sensitive potassium channel coexpressed with Ci-VSP increases at hyperpolarization and decreases at depolarization (8). This result cannot be reconciled with the known enzymatic activity of Ci-VSP, which would result in increased PI(4,5)P 2 levels, thereby resulting only in activation of the potassium channel. In addition, confocal imaging of pleckstrin homology domains (PHDs) fused to GFP as detectors of PI(4,5)P 2 and PI(3,4,5)P 3 , as well as electrophysiological measurements of potassium currents in Xenopus oocytes, showed th...
The ␣7 nAChR-selective partial agonist 3-(2,4-dimethoxybenzylidene)anabaseine (GTS-21) is more efficacious and potent for rat receptors than for human ␣7 receptors. Four single amino acid differences exist between human and rat ␣7 in the agonist binding site, two in the C loop, and one each in the E and F loops. Reciprocal mutations were made in these three domains and evaluated in Xenopus laevis oocytes. Mutations in the C and F loops significantly increased the efficacy of GTS-21 for the human receptor mutants but not to the level of the wild-type, and reciprocal mutations in rat ␣7 did not decrease responses to GTS-21. Whereas mutations in the E loop alone were without effect, the E-and F-loop mutations together increased GTS-21 efficacy and potency for human receptors, but the EF mutations in the rat receptors decreased the GTS-21 potency without changing the efficacy. The only mutants that showed a full reversal of the efficacy differences between human and rat ␣7 contained complete exchange of all four sites in the C, E, and F loops or just the sites in the C and F loops. However, the reversal of the potency ratio seen with the EF mutants was not evident in the CEF mutants. Our data therefore indicate that the pharmacological differences between rat and human ␣7 receptors are caused by reciprocal differences in sites within and around the binding site. Specific features in the agonist molecule itself are also identified that interact with these structural features of the receptor agonist binding site.A crucial assumption for the translation of preclinical research from animal studies to human therapeutics is that receptor pharmacology will be consistent between species. That is, drugs shown to be useful based on their ability to work in animal (rodent) models would also have similar activity on human forms of the receptors. The neuronal ␣7-type nicotinic acetylcholine receptor (nAChR) has been identified as a potential target for the treatment of Alzheimer's disease (Lindstrom, 1997), and 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21; also called DMXBA), which selectively targets this receptor, has been shown to improve learning and memory in animal models of cholinergic hypofunction (Kem, 2000). This ␣7-selective partial agonist has also been shown to prevent the death of differentiated PC-12 cells that occurs after nerve growth factor removal and the death of cultured primary neurons that occurs after high levels of NMDA receptor activation (Martin et al., 1994;Shimohama et al., 1998). It is interesting that although GTS-21 was able to protect PC-12 cells from the cytotoxic effects of amyloid peptide exposure, it was not able to protect human-derived SK-N-SH cells from the same cytotoxic stress, although the GTS-21 4-hydroxy metabolite, 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4-OH-GTS-21), was cytoprotective in the same assay (Meyer et al., 1998a). A likely explanation for these observed differences in cytoprotective activity came from the observation that GTS-21 was far less efficacious ...
Homomeric ␣7 and heteromeric ␣42 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in ␣7 receptors for which the ␣7-selective agonist 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in ␣4 receptors co-expressed with 2 resulted in ␣42 receptors for which ACh potency was relatively unchanged, although the efficacy of the ␣7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in ␣7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both ␣7 and ␣4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the ␣4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.
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