Robust photoregulation of GABA(A) receptors by allosteric modulation with a propofol analogue.

Photochemical switches represent a powerful method for improving pharmacological therapies and controlling cellular physiology. Here we report the photoregulation of GABA(A) receptors (GABA(A)Rs) by a derivative of propofol (2,6-diisopropylphenol), a GABA(A)R allosteric modulator, which we have modified to contain photoisomerizable azobenzene. Using α(1)β(2)γ(2) GABA(A)Rs expressed in Xenopus laevis oocytes and native GABA(A)Rs of isolated retinal ganglion cells, we show that the trans-azobenzene isomer of the new compound (trans-MPC088), generated by visible light (wavelengths ~440 nm), potentiates the γ-aminobutyric acid-elicited response and, at higher concentrations, directly activates the receptors. cis-MPC088, generated from trans-MPC088 by ultraviolet light (~365 nm), produces little, if any, receptor potentiation/activation. In cerebellar slices, MPC088 co-applied with γ-aminobutyric acid affords bidirectional photomodulation of Purkinje cell membrane current and spike-firing rate. The findings demonstrate photocontrol of GABA(A)Rs by an allosteric ligand, and open new avenues for fundamental and clinically oriented research on GABA(A)Rs, a major class of neurotransmitter receptors in the central nervous system

Molecular determinants of ATP binding to P2X2 ion channels.

ATP, in addition to its myriad roles in intracellular processes, is an extracellular signaling molecule. ATP serves as a neurotransmitter by binding to and opening ion channels known as P2X receptors. P2X receptors assemble as trimers of subunits. It is unclear if ATP binding occurs between or within subunits and if three ATP are required to bind the receptor before the channel opens. To address these questions, we adopted two approaches, both based on introducing mutations that disrupt ATP binding at one or two of the three ATP binding sites of P2X2 receptors. Some of these mutations (K69A and K308A) were sufficiently severe that homotrimers bearing them were non-responsive to ATP. First, we characterized in Xenopus laevis oocytes several concatamers in which the open reading frames for three subunits were joined together to form a single coding unit with binding site mutations introduced at various positions. We obtained evidence that these concatamers could form a subpopulation of cross-assembly side-products, which limited the conclusions we could draw. Second, we co-expressed subunits encoding ATP binding mutants either together or with subunits which respond normally to ATP. From these experiments we concluded that receptors with one wild type and two mutant binding sites could respond to ATP and that binding is between receptor subunits. In a final set of experiments, we used two cysteine mutants, one at a critical ATP binding residue (K69C) and another in a neighboring but non-binding residue (I67C). K69C did not give rise to detectable ATP responses, but treatment with Alexa Fluor 546 C5-maleimide (AM546), a thiol-reactive drug, caused the channels to be constitutively open, and to respond to the allosteric modulators zinc and acidic pH in the absence of exogenous ATP. Therefore, this mutant could serve as a specific biosensor of physiological zinc and pH. In contrast, AM546 caused the responses of I67C channels to decline to less than 10% of their initial amplitude. Additional experiments provided evidence that ATP binding was abolished at these receptors, therefore this mutant will be useful in concatamer and co-expression based experiments to explore the requirement for ATP binding in receptor activation.Ph.D.Biological SciencesBiophysicsMolecular biologyNeurosciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127049/2/3328807.pd

AM546 activated the K69C receptor.

<p>A–B: Sample traces from oocytes expressing either wild type rP2X2 (WT) or K69C as they were exposed to 50 µM AM546 for 5 minutes and then washed with recording solution. The horizontal dotted lines indicate the pretreatment holding current levels. The vertical dashed lines to the right of each trace indicate the difference in holding current 2 min after washout from the holding current before exposure to the drug. For wild type the change was so small that the whiskers obscure the amplitude of the dashed line. C: AM546 treatment caused K69C expressing oocytes to become responsive to zinc and acidic pH in the absence of exogenous ATP, but had no effect on responses of wild type receptors. Note the change in holding current after AM546 treatment in K69C expressing oocytes but not wild type oocytes (vertical dashed lines to the right of the traces). D–E: Quantitative characterization of a series of oocytes studied as in C, as well as control oocytes exposed to the vehicle alone (0.5% DMSO) (N = 4–8). Asterisks indicate a statistically significant difference from the DMSO control tested on the same construct.</p

Covalent Modification of Mutant Rat P2X2 Receptors with a Thiol-Reactive Fluorophore Allows Channel Activation by Zinc or Acidic pH without ATP

<div><p>Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C₅-maleimide (AM546). <em>Xenopus</em> oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM) or acidic external solution (pH 6.5) elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.</p> </div

Concentration-response relations for the effects of zinc and pH.

<p>A: Effect of increasing zinc concentration on the currents of an oocyte expressing wild type rP2X2 in the continuous presence of 5 µM ATP. There was already a substantial inward current at 5 µM ATP with 5 µM zinc, but for quantifying the magnitude of the effect for a series of similar oocytes, the current with 5 µM zinc was defined as 0, and changes from this level are shown in the trace and the accompanying graph in C. B: Effect of increasing zinc concentration, in the absence of exogenous ATP, on the currents of an oocyte expressing K69C after treatment with AM546. The current with 5 µM zinc was defined as 0. C: Quantification of a series of oocytes studied as in A and B. The data points for wild type rP2X2 (filled circles) are scaled by the left y-axis and the data points for K69C (open circles) are scaled by the right y-axis (N = 5). D: Effect of increasing proton concentration on the currents of an oocyte expressing wild type rP2X2 in the continuous presence of 5 µM ATP. The currents at pH 8.5 were defined as 0. E: Effect of increasing proton concentration, in the absence of exogenous ATP, on the currents of an oocyte expressing K69C after treatment with AM546. The currents at pH 8.5 were defined as 0. F: Quantification of a series of oocytes studied as in D and E. The data points for wild type rP2X2 (filled circles) are scaled by the left y-axis and the data points for K69C (open circles) are scaled by the right y-axis (wild type, N = 7; K69C, N = 3).</p

Effects of AM546 on the K69C/H319K double mutant.

<p>A: Responses to acidic pH and zinc before and after AM546 treatment. The dashed lines indicate the 0 current level, and indicate that there was a large inward holding current prior to any treatment. B: Average responses for a series of cells studied as in A. (N = 16 for holding current, 8 for zinc and 4 for pH 6.5). In contrast to the other figures, inward currents are plotted as downward deflections, to emphasize the different signs of the responses to zinc and acidic pH in this mutant. C: ATP concentration-response relations before and after AM546 treatment (N = 4).</p

Responses of AM546-treated K69C receptors to suramin.

<p>A: Effect of suramin on an oocyte expressing wild type rP2X2 when applied without exogenous ATP. The horizontal dashed line indicates the 0 current level. B: Correlation between the initial holding current of oocytes expressing wild type rP2X2 and the amplitude of the outward current in response to suramin (20 µM) or apyrase (1 mg/ml) C: Average amplitude of the outward current in response to suramin, apyrase, or recording solution alone applied from a separate barrel of the solution switcher (control). D: Effect of suramin on an oocyte expressing K69C before AM546 (thin gray trace) and after AM546 (thick black trace). The two traces were <i>not</i> baseline corrected to indicate the drop in holding current caused by AM546 (vertical dashed line). E: Concentration-response plots of suramin on wild type rP2X2 and AM546-treated K69C receptors (N = 6). F: Response of a K69C oocyte to 200 µM suramin after treatment with AM546 and then retested after exposure to MTSES. G: Results of a series of experiments as in F. The “AM546 after MTSES” group represents a control to verify that the dose of MTSES given to the “MTSES after AM546” experimental group was sufficient to occupy all free cysteines.</p

Structural features likely contributing to activation of AM546 modified K69C channels by zinc or pH.

<p>A: Structure of ATP folded as it sits in the binding site of zebrafish P2X4.1, and structure of AM546 with the linker folded back upon itself. B: Structure of P2X4.1 in the closed and open (ATP-bound) states. Only two of the three subunits are illustrated. Residues that bind ATP are shown in ball and stick format. The gray boxes superimposed on the closed state indicate domains of the receptor named according to the dolphin model of Kawate and Gouaux <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047147#pone.0047147-Kawate1" target="_blank">[32]</a>. Left: The closed state structure of zP2X4.1 (PDB 4DW0) with positions homologous to key residues of rP2X2 colored. Orange indicates the location of the histidines of the potentiating zinc binding site of rP2X2, which are P125 (cyan subunit) and H219 (pink subunit) in zP2X4.1. Beige is the location of F327 (of the cyan subunit), which is homologous to rP2X2 H319. Blue (L64) and purple (P199) indicate the residues (of the pink subunit) that bind to F327 in the closed state. Right: The ATP bound open state structure of zP2X4.1 (PDB 4DW1). Residues are colored as in A. The ATP at the interface between the two illustrated subunits is also shown (green). Note that the side group of H219 has been rotated from its position in 4DW1 to emphasize the close apposition of the orange histidines that is possible in rP2X2 when zinc is present.</p

Effect of other Alexa maleimides on K69C responses.

<p>In these experiments K69C expressing oocytes were first exposed to the test compound (50 µM, 5 minutes), then tested for responses to zinc, pH 6.5 and ATP, then exposed to the challenge compound (50 µM, 5 minutes). The purpose of the challenge compound application was to verify that the test compound had successfully bound to the K69C receptors. If it had not, then responses after the challenge compound would have been similar to the challenge compound given alone. Data for the MTSEA control are also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047147#pone-0047147-g001" target="_blank">Figure 1</a> and data for the AM546 control are also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047147#pone-0047147-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047147#pone-0047147-g006" target="_blank">Figure 6B–C</a>.</p