Caffeine inhibition of ionotropic glycine receptors

Abstract

We found that caffeine is a structural analogue of strychnine and a competitive antagonist at ionotropic glycine receptors (GlyRs). Docking simulations indicate that caffeine and strychnine may bind to similar sites at the GlyR. The R131A GlyR mutation, which reduces strychnine antagonism without suppressing activation by glycine, also reduces caffeine antagonism. GlyR subtypes have differing caffeine sensitivity. Tested against the EC(50) of each GlyR subtype, the order of caffeine potency (IC(50)) is: alpha2beta (248 +/- 32 microm) alpha3beta (255 +/- 16 microm) > alpha4beta (517 +/- 50 microm) > alpha1beta(837 +/- 132 microm). However, because the alpha3beta GlyR is more than 3-fold less sensitive to glycine than any of the other GlyR subtypes, this receptor is most effectively blocked by caffeine. The glycine dose-response curves and the effects of caffeine indicate that amphibian retinal ganglion cells do not express a plethora of GlyR subtypes and are dominated by the alpha1beta GlyR. Comparing the effects of caffeine on glycinergic spontaneous and evoked IPSCs indicates that evoked release elevates the glycine concentration at some synapses whereas summation elicits evoked IPSCs at other synapses. Caffeine serves to identify the pharmacophore of strychnine and produces near-complete inhibition of glycine receptors at concentrations commonly employed to stimulate ryanodine receptors.

Figure 1. Caffeine suppresses exogenous glycine-activated current on retinal ganglion cells (RGCs)

A, caffeine suppressed the response to 100 μm glycine (dark bars above current traces) in a retinal ganglion cell in a dose-dependent manner. B, 5 mm glycine overcame the caffeine block. C, dose–response curve of caffeine suppression of 100 μm glycine-activated current (IC₅₀= 1.67 ± 0.23 mm, Hill coefficient = 1.3 ± 0.2, n= 9). D and E, the currents produced by100 μm glycine were inhibited by 2 mm theophylline (D) or 2 mm theobromine (E). Ganglion cells in the retinal slice preparation were voltage clamped at 0 mV.

Figure 2. Structural similarities between caffeine and strychnine

A, electrostatic potentials (EPS) and B, hydrogen bonding maps of caffeine (left) and strychnine (right). Both molecules have three electronegative (blue) atoms (colour scale: red, most positive; purple, most negative) that are also hydrogen acceptors (colour scale: blue, high H acceptor density; red, high H donor density). C, alignment of caffeine (green) and strychnine (hydrogen atoms omitted) based on closest distance between three atom pairs (O2, O6, N9 on caffeine and O10, O24, N19 on strychnine). D, simulated docking of strychnine (blue) and caffeine (red) to an α1 dimer. The GlyR ligand binding domain model was obtained through homology modelling with nicotinic AChR ligand binding domain (see Methods). Using this model, caffeine and strychnine were docked to an overlapping binding region on the interface between two α1 subunits.

Figure 3. The R131A GlyR mutation decreases caffeine sensitivity of the α1 GlyR

HEK293 cells expressing rat homomeric wild type α1 (A) or mutant α1 R131A (B) GlyRs were held at −20 mV to record glycine-activated chloride currents. The cells were exposed to 60 μm (EC₅₀) glycine in the presence or absence of various concentrations of caffeine. The mutant GlyR (n= 7) caffeine IC₅₀ was over 10 times higher than wild type (n= 4).

Figure 4. Caffeine blocks GlyRs at an extracellular site

Exogenous 100 μm glycine (indicated by bar above current trace)-activated currents recorded from retinal ganglion cells held at 0 mV. A, responses to glycine alone (black trace) or glycine in presence of 10 μm ryanodine (grey trace). Cells were pretreated with 10 μm ryanodine for 40 s before co-application with glycine. B, the pipette solution contained 10 mm BAPTA. After allowing several minutes for dialysis, neurons were exposed to glycine alone (black trace) or together with 10 mm caffeine (grey trace). C, currents elicited by glycine alone (black trace) or in the presence of 100 μm IBMX (grey trace). D, the pipette solution contained 10 mm caffeine. After several minutes of dialysis, neurons were exposed to glycine alone (left), together with 10 mm extracellular caffeine (middle), then glycine alone after removal of caffeine (right).

Figure 5. Caffeine inhibition of GlyR subtypes

HEK293 cells expressing rat heteromeric αβ GlyRs were held at −20 mV to record chloride currents. The effects of various concentrations of caffeine were tested against the EC₅₀ concentration of glycine for each GlyR subtype: A, 60 μm glycine for α1β (n= 8); B, 100 μm glycine for α2β (n= 7); C, 340 μm glycine for α3β (n= 7); and D, 70 μm glycine for α4β (n= 10). Data were fitted to the Hill equation (see Methods).

Figure 6. Caffeine inhibits glycine responses in α3β GlyRs

Various concentrations of glycine with or without 300 μm caffeine were applied to HEK293 cells expressing α3β GlyRs. The glycine EC₅₀ concentration was increased from 338 ± 15 μm (n= 9) to 497 ± 33 by 300 μm caffeine (n= 9).

Figure 7. Caffeine suppression of spontaneous and light-evoked glycinergic IPSCs on retinal ganglion cells

Retinal ganglion cells were held at 0 mV to record IPSCs. GABAergic inputs were blocked by SR95531 (5 μm). A, decreases in amplitude of both spontaneous IPSCs and light-evoked IPSCs were noted in the presence of 200 μm caffeine. 2 mm caffeine completely suppressed all glycinergic IPSCs. B, 5 mm caffeine blocked spontaneous but not light-evoked IPSCs. C and D, spontaneous glycinergic IPSCs were measured before and during the application of 500 μm caffeine. C, caffeine did not alter the inter-event interval between spontaneous events. D, caffeine did reduce the amplitude of spontaneous events. E, a dose–response curve showing the effect of various caffeine concentrations on the peak light-evoked glycinergic IPSCs recorded in ganglion cells. Cells were held at 0 mV and GABAergic responses were blocked with 5 μm SR95531. A Hill plot to the data yielded a caffeine IC₅₀ of 1.65 ± 0.22 mm (n= 12). F, a ganglion cell was held at −70 mV to isolate the light-evoked EPSC and 50 μm picrotoxinin and 10 μm strychnine were co-applied to eliminate inhibitory currents. Light-evoked EPSCs were recorded in the absence (black trace) or presence of 10 mm caffeine (grey trace).