Abstract
Purpose: :
Glycine receptors (GlyRs) can be formed from among four different alpha subunits. In the pentameric receptor, two alpha subunits couple with three beta units (only one known type). We have developed a GlyR pharmacology in HEK293 cells and related it to retinal GlyRs. However, published reports describe retinal GlyR properties uncorrelated with expression system data. We addressed this discrepancy.
Methods: :
Drug effects were tested in isolated rat retinal third order neurons and GlyR-transfected HEK 293 cells using whole cell patch recordings.
Results: :
In HEK293 cells, the α3β GlyRs are very insensitive to glycine (EC50 = 396µM). This compares with EC50s for α1β, α2β, and α4β of 60 µM, 100 µM, and 70 µM, respectively. All GlyR dose response-curves had a Hill coefficient of ~ 2. In contrast, rat retinal neurons have an EC50 of 295µM and a Hill coefficient = ~ 1. The apparent high EC50 and low Hill coefficient are artifacts caused by the medley of GlyR subtypes in single retinal neurons. A multifit analysis of retinal GlyR dose-response curves revealed the presence of two main GlyR subtypes. This could be tested because α3β GlyR is extremely sensitive to picrotoxin (PTX, IC50 = 12 µM). In the presence of PTX, the second curve component was suppressed and the dose-responses in retina and α1β GlyR in HEK 293 cells were now similar. The response of mixtures of α1β and α3β GlyRs in HEK293 cells supported the multi-subunit interpretation. In HEK293 cells, coexpression of α1 and α3 GlyRs results in homomeric and heteromeric receptors. However, retinal neurons do no exhibit response properties indicative of heteromeric receptors.
Conclusions: :
The elevated EC50 and low Hill coefficient detected in retinal neurons is due to multiple GlyR subtypes within single retinal neurons. The α3 GlyR acts to both elevate the EC50 and reduce the sensitivity to glycine. This improves the dynamic range of inhibition. Comparing results in HEK293 and retina indicates that receptor sorting in retina is specialized to ensure that individual receptors incorporate only one type of α subunit. Thus, in retina there are likely to be high affinity and low affinity glycine synapses.
Keywords: inhibitory neurotransmitters • inhibitory receptors • receptors: pharmacology/physiology