Rescue of homeostatic regulation of striatal excitability and locomotor activity in a mouse model of Huntington's disease.

We describe a fast activity-dependent homeostatic regulation of intrinsic excitability of identified neurons in mouse dorsal striatum, the striatal output neurons. It can be induced by brief bursts of activity, is expressed on a time scale of seconds, limits repetitive firing, and can convert regular firing patterns to irregular ones. We show it is due to progressive recruitment of the KCNQ2/3 channels that generate the M current. This homeostatic mechanism is significantly reduced in striatal output neurons of the R6/2 transgenic mouse model of Huntington's disease, at an age when the neurons are hyperactive in vivo and the mice begin to exhibit locomotor impairment. Furthermore, it can be rescued by bath perfusion with retigabine, a KCNQ channel activator, and chronic treatment improves locomotor performance. Thus, M-current dysfunction may contribute to the hyperactivity and network dysregulation characteristic of this neurodegenerative disease, and KCNQ2/3 channel regulation may be a target for therapeutic intervention.

Covalent modification of mutant rat P2X2 receptors with a thiol-reactive fluorophore allows channel activation by zinc or acidic pH without ATP.

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 C(5)-maleimide (AM546). Xenopus 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.

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)ß(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.

GABAA receptors increase excitability and conduction velocity of cerebellar parallel fiber axons.

In the adult mammalian brain, GABA(A) receptors (GABA(A)Rs) are responsible for the predominant forms of synaptic inhibition, but these receptors can excite neurons when the chloride equilibrium potential (E(Cl)) is depolarized. In many mature neurons, GABA(A)Rs are found on presynaptic terminals where they exert depolarizing effects. To understand whether excitatory GABA action affects axonal function, we used transverse cerebellar slices to measure the effects of photolysis of caged GABA on the initiation and propagation of compound parallel fiber (PF) action potentials (APs). Photolysis of caged GABA increased the amplitude and conduction velocity of PF APs; GABA reuptake blockers and a positive modulator of GABA(A)Rs enhanced these effects. In contrast, a modulator selective for δ-subunit-containing GABA(A)Rs did not enhance these effects and responsiveness remained in δ(-/-) mice, arguing that δ-subunit-containing GABA(A)Rs are not required. Synaptically released GABA also increased PF excitability, indicating that the mechanism is engaged by physiological signals. A Hodgkin-Huxley-style compartmental model of the PF axon and granule cell body was constructed, and this model recapitulated the GABA-dependent decrease in AP threshold and the increase in conduction velocity, features that were sensitive to E(Cl) and to the voltage dependence of sodium channel inactivation. The model also predicts that axonal GABA(A)Rs could affect orthodromic spike initiation. We conclude that GABA acting on cerebellar PFs facilitates both spike generation and propagation, allowing axons of granule cells to passively integrate signals from inhibitory interneurons and influence information flow in the input layer to the cerebellar cortex.

An intersubunit zinc binding site in rat P2X2 receptors.

P2X receptors are ATP-gated ion channels made up of three similar or identical subunits. It is unknown whether ligand binding is intersubunit or intrasubunit, either for agonists or for allosteric modulators. Zinc binds to rat P2X2 receptors and acts as an allosteric modulator, potentiating channel opening. To probe the location of this zinc binding site, P2X2 receptors bearing mutations of the histidines at positions 120 and 213 were expressed in Xenopus oocytes. Studies of H120C and H213C mutants produced five lines of evidence consistent with the hypothesis that the residues in these positions bind zinc. Mixing of subunits containing the H120A or H213A mutation generated receptors that showed zinc potentiation, even though neither of these mutant receptors showed zinc potentiation on its own. Furthermore, expression of trimeric concatamers with His --> Ala mutations at some but not all six positions showed that zinc potentiation correlated with the number of intersubunit histidine pairs. These results indicate that zinc potentiation requires an interaction across a subunit interface. Expression of the H120C/H213C double mutant resulted in the formation of ectopic disulfide bonds that could be detected by changes in the physiological properties of the receptors after treatment with reducing and oxidizing agents. Immunoblot analysis of H120C/H213C protein separated under nonreducing conditions demonstrated that the ectopic bonds were between adjacent subunits. Taken together, these data indicate that His120 and His213 sit close to each other across the interface between subunits and are likely to be key components of the zinc binding site in P2X2 receptors.