Characteristics of glycine receptors expressed by embryonic rat brain mRNAs

Abstract

A study was made of glycine (Gly) and gamma-aminobutyric acid (GABA) receptors expressed in Xenopus oocytes injected with rat mRNAs isolated from the encephalon, midbrain, and brainstem of 18-day-old rat embryos. In oocytes injected with encephalon, midbrain, or brainstem mRNAs, the Gly-current amplitudes (membrane current elicited by Gly; 1 mM Gly) were respectively 115 +/- 35, 346 +/- 28, and 389 +/- 22 nA, whereas the GABA-currents (1 mM GABA) were all < or =40 nA. Moreover, the Gly-currents desensitized faster in oocytes injected with encephalon or brainstem mRNAs. The EC(50) for Gly was 611 +/- 77 microM for encephalon, 661 +/- 28 microM for midbrain, and 506 +/- 18 microM for brainstem mRNA-injected oocytes, and the corresponding Hill coefficients were all approximately 2. Strychnine inhibited all of the Gly-currents, with an IC(50) of 56 +/- 3 nM for encephalon, 97 +/- 4 nM for midbrain, and 72 +/- 4 nM for brainstem mRNAs. During repetitive Gly applications, the Gly-currents were potentiated by 1.6-fold for encephalon, 2.1-fold for midbrain, and 1.3-fold for brainstem RNA-injected oocytes. Raising the extracellular Ca(2+) concentration significantly increased the Gly-currents in oocytes injected with midbrain and brainstem mRNAs. Reverse transcription-PCR studies showed differences in the Gly receptor (GlyR) alpha-subunits expressed, whereas the beta-subunit was present in all three types of mRNA. These results indicate differential expression of GlyR mRNAs in the brain areas examined, and these mRNAs lead to the expression of GlyRs that have different properties. The modulation of GlyRs by Ca(2+) could play important functions during brain development.

Figure 1

Expression of Gly and GABA receptors by embryonic mRNAs. The columns represent the amplitude of membrane currents elicited by Gly or GABA (both 1 mM) in oocytes injected with mRNA from 18-day-old rat embryo encephalon, midbrain, or brainstem. (Inset) Representative Gly-currents. For this and subsequent figures, the downward deflections correspond to inward currents, the membrane potential was held at −60 mV (unless otherwise indicated), and Gly was applied as indicated by a continuous line and by brief depolarizing pulses. Each column represents the mean + SEM. The number of oocytes studied (from five donors) were 26, 43, and 18 (Gly-currents), and 26, 10, and 36 (GABA-currents) for encephalon, midbrain, and brainstem mRNAs, respectively.

Figure 2

Gly dose–response relationships. Current amplitude as a function of Gly concentration, from oocytes injected with encephalon, midbrain, or brainstem mRNAs. Responses were normalized to the current at 3 mM. Continuous lines represent least-squares fits to the Hill equation. Each point shows the mean ± SEM for five oocytes from three different donors in midbrain and brainstem, and two donors for encephalon. (Inset) Typical currents elicited by 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, and 1.0 (midbrain), and by 0.1, 0.2, 0.3, 0.4, 0.5, and 0.75 mM Gly (brainstem).

Figure 3

Gly-current–voltage relationship. Current-to-voltage relationship from one oocyte injected with brainstem mRNA obtained by applying brief voltage steps, from −140 to 40 mV, in the absence and presence of 2 mM Gly and subtracting the passive membrane currents in the absence of Gly. (Inset) Representative Gly-currents in another oocyte. Gly (2 mM) was applied every 10 min at the indicated membrane potentials.

Figure 4

Potentiation of Gly-currents. Amplitudes of repetitive Gly-currents in oocytes injected with encephalon (n = 2), midbrain (n = 4), or brainstem (n = 8) mRNAs. Gly (1 mM) was applied for 2 min at about 10 min intervals. Time 0 indicates the first application; potentiation was greater for midbrain than for encephalon or brainstem. (Inset) Examples of the first and fourth Gly-currents from an oocyte injected with brainstem mRNA.

Figure 5

Gly current modulation by calcium. The columns represent the mean + SEM (n = 3) of Gly-currents elicited by 1 mM Gly in the presence of different Ca²⁺ concentrations. (Inset) Currents elicited by 1 mM Gly in normal Ringer's solution, and in the presence of high Ca²⁺ (12 mM) in an oocyte injected with midbrain mRNA.

Figure 6

Inhibition of Gly-currents by strychnine. Gly-current amplitudes normalized to the current in the absence of strychnine. Continuous lines represent least squares fits to the Hill equation. Each point shows the mean ± SEM for three oocytes injected with mRNAs from midbrain and brainstem, and from one oocyte injected with mRNA from encephalon. The oocytes were from three different donors. (Inset) Typical currents elicited by 1 mM Gly and inhibited by strychnine in an oocyte injected with midbrain mRNA.

Figure 7

Distribution of GlyR-subunit transcripts. (A) The lanes show the GlyR subunits present in encephalon, midbrain, and brainstem mRNAs amplified by RT-PCR. Subunits: α1-, α2-, α3-, and β- as well as negative (c⁻, water) and positive (c⁺, glyceraldehyde-3-phosphate dehydrogenase) controls. (B) Semiquantitative signals in 1.5% agarose gel; the signals were designated weak, +; moderate, ++; and intense, +++. ND, not detected. Note that the α1- and β-subunits were amplified from all mRNAs, and that the α2- and α3-subunits were present in encephalon and brainstem but not in the midbrain.