Andrew J. Boileau

Effects of γ2S subunit incorporation on GABAA receptor macroscopic kinetics

Abstract GABAA receptors, the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are heteropentameric proteins. We are interested in understanding the contribution of the γ subunit to the kinetic properties of GABAA receptors. Studies in Xenopus oocytes have suggested that co-expression of α1, β2, and γ2S subunits results in the formation of both αβ and αβγ receptors ( Boileau et al., 2002a , Boileau et al., 1998 ). Here, we have used an excess of the γ2S subunit in transfections of HEK293 cells to bias expression toward αβγ-containing receptors. Using rapid application and whole cell patch clamp techniques, we found that incorporation of the γ subunit eliminated the rapid phases of desensitization and accelerated deactivation, consistent with a proposed role of desensitization in slowing deactivation. In addition, αβγ receptors had an increased GABA EC50, reduced sensitivity to block by Zn2+, and did not display outward rectification as compared to αβ receptors.

Tandem subunits effectively constrain GABAA receptor stoichiometry and recapitulate receptor kinetics but are insensitive to GABAA receptor-associated protein.

GABAergic synapses likely contain multiple GABAA receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of α, β, and γ subunits can be confounded by receptors expressed from α and β subunits alone. To produce recombinant GABAA receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a “tandem” α1 subunit linked to a β2 subunit. Cotransfection of the γ2 subunit with αβ-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the γ subunit with nonlinked α1 and β2 subunits. Similarly, expression of α subunits together with αβ-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of α with β subunits. In this first description of tandem GABAA subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of αβ-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of α1β2 and α1β2γ2 receptors. We used this method to test whether the accessory protein GABAA receptor-associated protein (GABARAP) alters GABAA receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in α1β2γ2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of αβγ receptors.

Optimized expression vector for ion channel studies in Xenopus oocytes and mammalian cells using alfalfa mosaic virus

Plasmid vectors used for mammalian expression or for in vitro cRNA translation can differ substantially and are rarely cross-compatible. To make comparisons between mammalian and Xenopus oocyte expression systems, it would be advantageous to use a single vector without the need for shuttle vectors or subcloning. We have designed such a vector, designated pUNIV for universal, with elements that will allow for in vitro or ex vivo expression in multiple cell types. We tested the expression of pUNIV-based cDNA cassettes using enhanced green fluorescent protein and two forms of the type A γ-aminobutyric acid receptor (GABAAR) and compared pUNIV to vectors optimized for expression in either Xenopus oocytes or mammalian cells. In HEK293 cells, radioligand binding was robust, and patch clamp experiments showed that subtle macroscopic GABAAR kinetics were indistinguishable from our previous results. In Xenopus oocytes, agonist median effective concentration measurements matched previous work using a vector optimized for oocyte expression. Furthermore, we found that expression using pUNIV was significantly enhanced in oocytes and was remarkably long-lasting in both systems.

A GABAA Receptor α1 Subunit Tagged with Green Fluorescent Protein Requires a β Subunit for Functional Surface Expression

γ-Aminobutyric acid, type A (GABAA) receptors, the major inhibitory neurotransmitter receptors in the central nervous system, are heteropentameric proteins assembled from distinct subunit classes with multiple subtypes, α(1–6), β(1–4), γ(1–3), δ(1), and ε(1). To examine the process of receptor assembly and targeting, we tagged the carboxyl terminus of the GABAA receptor α1 subunit with red-shifted enhanced green fluorescent protein (EGFP). Xenopus oocytes were injected with cRNA of this fusion protein, α1-EGFP, alone or in combination with cRNA of GABAA receptor β2, γ2, or β2+γ2 subunits. Within 72 h after injection, EGFP fluorescence was visible in all fusion protein-injected cells. The fluorescence was associated with the plasmalemma only when the β2 subunit was co-injected with α1-EGFP. Texas Red-conjugated immunolabeling of EGFP on nonpermeabilized cells demonstrated that EGFP was localized extracellularly. Hence, the COOH terminus of the α1subunit is extracellular. Two-electrode voltage clamp of α1-EGFPβ2- and α1-EGFPβ2γ2-injected oocytes demonstrates that these cells express functional receptors, with EC50 values for GABA and diazepam similar to wild-type receptors. Thus, a COOH-terminal tag of the α1 subunit appears to be functionally silent, providing a useful marker for studies of GABAA receptor expression, assembly, transport, targeting, and clustering. Moreover, the β2 subunit is required for receptor assembly and surface expression.

Transformation of Paramecium tetraurelia by Electroporation or Particle Bombardment

ABSTRACT Methods for mass transformation of Paramecium tetraurelia were established using plasmids bearing neomycin‐resistance or calmodulin gene fragments. Phenotypic and molecular analyses showed that, although variable, up to 5% transformation can be achieved by electroporation. Concentrations of divalent cations Ca2‐ and Mg2+ in the electroporation medium were crucial for efficient transformation. Strong neomycin‐resistance transformation using bioballistic particle bombardment with gold particles was observed. For both methods, hybridization to transformant DNA revealed plasmid signals consistent with macronuclear transformation and correlated with transformed phenotypes. Complementation of a known calmodulin gene mutation was also achieved by mass transformation. Possible sources of variation and the general utility of these methods are discussed.

The Short Splice Variant of the γ2 Subunit Acts as an External Modulator of GABAA Receptor Function

GABAA receptors (GABAARs) regulate the majority of fast inhibition in the mammalian brain and are the target for multiple drug types, including sleep aids, anti-anxiety medication, anesthetics, alcohol, and neurosteroids. A variety of subunits, including the highly distributed γ2, allow for pharmacologic and kinetic differences in particular brain regions. The two common splice variants γ2S (short) and γ2L (long) show different patterns of regional distribution both in adult brain and during the course of development, but show few notable differences when incorporated into pentameric receptors. However, results presented here show that the γ2S variant can strongly affect both GABAAR pharmacology and kinetics by acting as an external modulator of fully formed receptors. Mutation of one serine residue can confer γ2S-like properties to γ2L subunits, and addition of a modified γ2 N-terminal polypeptide to the cell surface recapitulates the pharmacological effect. Thus, rather than incorporation of a separate accessory protein as with voltage-gated channels, this is an example of an ion channel using a common subunit for dual purposes. The modified receptor properties conferred by accessory γ2S have implications for understanding GABAAR pharmacology, receptor kinetics, stoichiometry, GABAergic signaling in the brain during development, and altered function in disease states such as epilepsy.