The short splice variant of the gamma 2 subunit acts as an external modulator of GABA(A) receptor function

Abstract

GABA(A) receptors (GABA(A)Rs) 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 gamma2, allow for pharmacologic and kinetic differences in particular brain regions. The two common splice variants gamma2S (short) and gamma2L (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 gamma2S variant can strongly affect both GABA(A)R pharmacology and kinetics by acting as an external modulator of fully formed receptors. Mutation of one serine residue can confer gamma2S-like properties to gamma2L subunits, and addition of a modified gamma2 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 gamma2S have implications for understanding GABA(A)R pharmacology, receptor kinetics, stoichiometry, GABAergic signaling in the brain during development, and altered function in disease states such as epilepsy.

Figure 1.

Diazepam potentiation and Zn²⁺ block for varying γ2S or γ2L transfection ratios. A, HEK293 cells expressing α1 plus β2 subunits. Left, Current traces from 500 ms, 3 μm GABA pulses (black) are superimposed with currents from the same outside-out patch exposed to a coapplication of 3 μm GABA plus 1 μm DZ (green) after equilibration in control solution plus 1 μm DZ. Potentiation (Pot) is reported as (I_{GABA + DZ}/I_GABA) − 1. Right, In the same patch, near-maximal current is elicited by 200 ms, 1 mm GABA pulses (black) and blocked by coapplication of 30 μm ZnCl₂ (red). Note that α1β2 receptors display significant Zn²⁺ block but no appreciable DZ potentiation. B, Traces for cells expressing α1 + β2 + γ2S (left) or γ2L (right) at indicated ratios. Note that three γ2L patches are shown from 1:1:0.1 ratio transfections to exhibit variability. Also included are traces for the α1β2γ2L receptors constrained by use of α1–β2 tandem receptors cotransfected with γ2L at a 2:1 ratio. Note that these receptors show similar high potentiation by DZ, minimal blockade by Zn²⁺, and minimal desensitization in 200 ms pulses (right) at 1 mm GABA, similar to α1β2γ2S receptors constrained by transfection at a 1:1:10 ratio. C, Paired Zn²⁺ block plotted against DZ potentiation for transfection ratios ranging from 1:1:0.01 to 1:1:10 for α1β2γ2, as well as tandem-constrained αβtan+γ (Boileau et al., 2005) and α1β2. Note that αβ receptors (open squares) show high zinc sensitivity and no potentiation, as do some of the patches from transfections with very low γ2 ratios, indicative of no γ2 subunits incorporated in that patch. The solid curve represents an interpolation between αβ and αβγ DZ potentiation and Zn²⁺ block values, modeled using EC₅₀, single-channel conductance, and open probability differences between the two receptor types (Boileau et al., 2005). The open circle lies on the model curve and has error bars corresponding to the 95% confidence intervals (CIs) for DZ potentiation and Zn²⁺ block (dashed lines). Note that many of the Zn²⁺ block values for transfections with γ2S ratios below 1:1:10 fall beneath the 95% confidence interval (C, left), whereas transfections varying γ2L ratios do not (C, right).

Figure 2.

γ2S subunits can also express independently, even in the presence of α1 and β2 subunits. A, Selected images from colocalization experiments performed on HEK293 cells transfected with α1β2γ2 cDNAs at 1:1:0.1, 1:1:1, and 1:1:10 ratios. Cells were labeled with 565 nm quantum dot-labeled antibodies (green) for α1 subunits and 655 nm (far red, pseudocolored red) for γ2 subunits (see Materials and Methods). Colocalized signal ranges from yellow–green to orange–red. Scale bar, 20 μm. B, Measurements of colocalization of γ2L or γ2S with α1 signal. At each transfection ratio, γ2S subunits were significantly different from γ2L subunits in the amount of red signal that was not colocalized with green (α1) signal. Data are mean ± SEM for n ≥ 50 cells per condition. *p < 0.01, **p < 0.001. C, Control measurements of α1 colocalization with γ2L or γ2S. At no transfection ratio does γ2S differ significantly from γ2L for this measurement.

Figure 3.

Mutations of serine 343 in γ2L subunits allow for rogue surface expression and anomalous Zn²⁺ blockade. A, Confocal images of cells cotransfected with EGFP and γ2L–S343D (serine to aspartate) or γ2L–S343V (serine to valine) are shown on the left. Scale bar, 20 μm. B, In separate experiments, excised patches from cells transfected with α1β2γ2L–S343D or α1β2γ2L–S343V (both at the transfection ratio 1:1:0.1) were tested for DZ potentiation and Zn²⁺ blockade, as in Figure 1. Note that both mutations cause γ2L to resemble γ2S.

Figure 4.

External application of truncated extracellular γ2 subunits reduces Zn²⁺ block in αβ receptors. A, Confocal images of cells cotransfected with α1 and β2 subunits and treated with immunopurified sticky proteins γ2N–K₆, α1N–K₆, β2N–K₆, or WGA linked to quantum dots. Cells were incubated in PBS for 60 min with immunopurified protein, washed several times, fixed, and treated with anti-FLAG antibody with 655 nm quantum dot-linked 2° antibody for the GABA_AR extracellular polypeptides. On the left are transmitted light images, and on the right are the pseudocolored far red images. Scale bar, 20 μm. B, Top, Traces from excised patches showing reduced Zn²⁺ blockade of α1β2 receptors incubated with the γ2 N-terminal protein γ2N–K₆. Traces with or without coapplied Zn²⁺ are the average of three traces from the same patch. Below are shown traces from α1β2 receptors incubated with control α1N–K₆, β2N–K₆, or quantum dot-linked WGA, none of which protected the receptors from Zn²⁺ block. C, Summary of Zn²⁺ block for α1β2 receptors (white bar), α1β2γ2 receptors (black; pooled α1β2γ2S 1:1:10, α1β2γ2L 1:1:10, αβtan+γ2S and αβtan+γ2L), and α1β2 receptors treated with γ2N–K₆ (red bar, average of 200 ng of protein per 12 mm coverslip in a 1 ml well) or controls α1N–K₆, β2N–K₆, or WGA (gray bars, average of 500 ng of protein per coverslip). *p < 0.001 significance of difference from α1β2 receptors; n.s., α1β2γ2 and α1β2 + γ2N-K₆ peptide not significantly different from one another. Inset, Depiction of the sticky γ2N–K₆ protein. D, Schematic of possible mechanisms for Zn²⁺ protective effect of γ2S on α1β2 receptors. i, The α1β2γ2 receptor has one putative external Zn²⁺ binding site (black hexagon) and one “weak” luminal binding site (gray hexagon). ii, The α1β2 receptor has two identical external Zn²⁺ binding sites and one internal site that may have higher affinity than in the case of α1β2γ2 receptors. i and ii patterned after Hosie et al. (2003). iii, Rogue γ2S subunits (as lone subunits or multimers of unknown number) may bind to α1β2 receptors and hinder binding of Zn²⁺ ions. iv, γ2S subunits may cause α1β2 receptors to cluster, thus reducing Zn²⁺ block of several receptors.

Figure 5.

Mixtures of α1β2 and α1β2γ2 receptors exhibit faster kinetics than either pure population but are altered by rogue γ2S subunits. A, Overlaid current traces from four patches each, from α1β2γ2S 1:1:10, 1:1:1, 1:1:0.5, 1:1:0.1, 1:1:0.03, and 1:1:0 (α1β2) transfections, in response to 200 ms pulses of 1 mm GABA. B, Expanded trace comparing fast desensitization in α1β2 receptors with α1β2γ2S 1:1:0.5. Note that the α1β2γ2 1:1:0.5 current is not only faster to desensitize but also faster to rise to peak. C, Plot of fast desensitization time constants (τ_fast) transfected with α1 and β2 subunits, with varying ratios of γ2S or γ2L subunits, wherein a fast component could be detected. Data are mean ± SEM for n = 17, 6, 13, 8, 3, 6, 7, and 10 patches (left to right). *p < 0.01 compared with α1β2 patches. D, Scatter plot of 10–90% rise times for the cells transfected with α1 and β2 subunits, with varying ratios of γ2L subunits. Note that ratios over a 100-fold range (1:1:0.1 to 1:1:10) give similar rise times for γ2L. E, Scatter plot of 10–90% rise times for αβγ2S transfections in varying ratios. Note that, as γ2S ratio is increased above 0.5, rise times are slowed. F, Models depicting fast desensitization differences between receptors and mixtures. i, α1β2γ2 receptors, possibly clustered, with trace showing no fast desensitization. In the case of γ2S only, rogue γ2S subunits may bind at the α/β interface to cause a slowing of the rise time (compare D with E at 1:1:1 and 1:1:10). ii, α1β2 receptor with fast desensitization. iii, Mixtures of αβ and αβγ receptors may cluster and change kinetic profiles to allow for the ultrafast (7 ms) desensitization time constant. Traces were culled from Figure 1.