Molecular Characterization of GABA-A Receptor Subunit Diversity within Major Peripheral Organs and Their Plasticity in Response to Early Life Psychosocial Stress

Abstract

Gamma aminobutyric acid (GABA) subtype A receptors (GABA_ARs) are integral membrane ion channels composed of five individual proteins or subunits. Up to 19 different GABA_AR subunits (α1-6, β1-3, γ1-3, δ, ε, θ, π, and ρ1-3) have been identified, resulting in anatomically, physiologically, and pharmacologically distinct multiple receptor subtypes, and therefore GABA-mediated inhibition, across the central nervous system (CNS). Additionally, GABA_AR-modulating drugs are important tools in clinical medicine, although their use is limited by adverse effects. While significant advances have been made in terms of characterizing the GABA_AR system within the brain, relatively less is known about the molecular phenotypes within the peripheral nervous system of major organ systems. This represents a potentially missed therapeutic opportunity in terms of utilizing or repurposing clinically available GABA_AR drugs, as well as promising research compounds discarded due to their poor CNS penetrance, for the treatment of peripheral disorders. In addition, a broader understanding of the peripheral GABA_AR subtype repertoires will contribute to the design of therapies which minimize peripheral side-effects when treating CNS disorders. We have recently provided a high resolution molecular and function characterization of the GABA_ARs within the enteric nervous system of the mouse colon. In this study, the aim was to determine the constituent GABA_AR subunit expression profiles of the mouse bladder, heart, liver, kidney, lung, and stomach, using reverse transcription polymerase chain reaction and western blotting with brain as control. The data indicate that while some subunits are expressed widely across various organs (α3-5), others are restricted to individual organs (γ2, only stomach). Furthermore, we demonstrate complex organ-specific developmental expression plasticity of the transporters which determine the chloride gradient within cells, and therefore whether GABA_AR activation has a depolarizing or hyperpolarizing effect. Finally, we demonstrate that prior exposure to early life psychosocial stress induces significant changes in peripheral GABA_AR subunit expression and chloride transporters, in an organ- and subunit-specific manner. Collectively, the data demonstrate the molecular diversity of the peripheral GABA_AR system and how this changes dynamically in response to life experience. This provides a molecular platform for functional analyses of the GABA-GABA_AR system in health, and in diseases affecting various peripheral organs.

Keywords: GABA; KCC2; NKCC1/2; chloride transporters; peripheral nervous system; psychosocial stress.

FIGURE 1

Diversity of GABA_AR subunit expression within various organs of the mouse. Representative gel electrophoresis images of mRNA transcripts for various GABA_AR subunits using RT-PCR and homogenates from the whole brain (Br), stomach (St), lung (Lu), bladder (Bl), kidney (Ki), heart (Hr), and liver (Li) obtained from adult male C57BL/6 mice. Corresponding amplicons of the same size bands to those obtained from the brain were consistently detected for various GABA_AR subunits within a number of peripheral organs. N = 3 animals.

FIGURE 2

Protein expression of the major GABA_AR α and γ subunits within various organs of the mouse. Representative western blots for GABA_AR α1–5 and γ2 subunits using tissue homogenates from whole brain (Br), stomach (St), lung (Lu), bladder (Bl), kidney (Ki), heart (Hr), and liver (Li) obtained from adult male C57BL/6 mice. Western blots for the different subunits in the peripheral organs corresponded to the sizes of those obtained from the brain. Note that blots for actin were run on separate gels due to the similar molecular weight of this protein compared to GABA_AR subunits. N = 3 animals.

FIGURE 3

Developmental changes in the mRNA expression level of the KCC2, NKCC1, and NKCC2 chloride ion transporters within various peripheral organs. Quantification of the mRNA expression levels of the potassium-chloride transporter member 5 (KCC2), Na–K–Cl cotransporter 1 (NKCC1), and Na–K–Cl cotransporter 2 (NKCC2) in tissue from mice aged postnatal day 6 (PND 6) and adulthood (PND 60), using qPCR. Within the (A) brain, (B) lung, and (C) heart, a significant increase in KCC2 was detected and a decrease in the NKCCs mRNA levels as the mice age. However, within the (D) stomach, the contrary is true. In addition, there is an increase in both KCC2 and NKCCs mRNA expression levels from PND 6 to 60 within the mouse bladder (E) and liver (F). Within the mouse (G) kidney, KCC2 mRNA is expressed at very low levels in compare to the NKCCs and aging induces a decrease in the expression of NKCCs. Data represent mean ± SEM; N = 5 animals. NS, not significant; ND, not detected. ^∗P < 0.05, Student’s unpaired t-test.

FIGURE 4

Early life stress (ELS)-induced changes in the level of GABA_AR subunit mRNA expression within various peripheral organs. Quantification of the ELS-induced changes in the expression levels of the (A) α1-GABA_AR subunit mRNA, (B) α2-GABA_AR subunit mRNA, (C) α3-GABA_AR subunit mRNA, (D) α4-GABA_AR subunit mRNA, (E) α5-GABA_AR subunit mRNA, and (F) γ2-GABA_AR subunit mRNA, within mouse stomach (St), lung (Lu), bladder (Bl), kidney (Ki), heart (Hr), and liver (Li) tissue, relative to the housekeeping gene GAPDH, using qPCR. Note that the mRNA expression of some subunits, relative to GAPDH, was not always detectable (ND) using qPCR. Data represent mean ± SEM; N = 5 animals. NS, not significant. Student’s unpaired t-test. ^∗P < 0.05.

FIGURE 5

Early life stress (ELS)-induced changes in the level of the potassium-chloride transporter member 5 (KCC2), Na–K–Cl cotransporter 1 (NKCC1), and Na–K–Cl cotransporter 2 (NKCC2) within various peripheral organs. Quantification of the mRNA expression levels of the KCC2, NKCC1, and NKCC2 in (A) brain, (B) stomach, (C) lung, (D) bladder, (E) kidney, (F) heart, and (G) liver tissue from adult mice (PND 60) exposed to ELS. Data represent mean ± SEM; N = 5 animals. NS, not significant. Student’s unpaired t-test. Note that the ELS induced significant changes in the mRNA expression of ion transporters specifically within the brain, stomach, lung, and bladder of the mouse. ^∗P < 0.05.