Salicylate, an aspirin metabolite, specifically inhibits the current mediated by glycine receptors containing alpha1-subunits

Abstract

Background and purpose: Aspirin or its metabolite sodium salicylate is widely prescribed and has many side effects. Previous studies suggest that targeting neuronal receptors/ion channels is one of the pathways by which salicylate causes side effects in the nervous system. The present study aimed to investigate the functional action of salicylate on glycine receptors at a molecular level.

Experimental approach: Whole-cell patch-clamp and site-directed mutagenesis were deployed to examine the effects of salicylate on the currents mediated by native glycine receptors in cultured neurones of rat inferior colliculus and by glycine receptors expressed in HEK293T cells.

Key results: Salicylate effectively inhibited the maximal current mediated by native glycine receptors without altering the EC(50) and the Hill coefficient, demonstrating a non-competitive action of salicylate. Only when applied simultaneously with glycine and extracellularly, could salicylate produce this antagonism. In HEK293T cells transfected with either alpha1-, alpha2-, alpha3-, alpha1beta-, alpha2beta- or alpha3beta-glycine receptors, salicylate only inhibited the current mediated by those receptors that contained the alpha1-subunit. A single site mutation of I240V in the alpha1-subunit abolished inhibition by salicylate.

Conclusions and implications: Salicylate is a non-competitive antagonist specifically on glycine receptors containing alpha1-subunits. This action critically involves the isoleucine-240 in the first transmembrane segment of the alpha1-subunit. Our findings may increase our understanding of the receptors involved in the side effects of salicylate on the central nervous system, such as seizures and tinnitus.

Figure 1

Salicylate (NaSal) inhibited the maximal I_Gly without significantly altering the EC₅₀ value and the Hill coefficient. (A) Sample currents recorded from one neurone of rat inferior colliculus induced by glycine (Gly) at 10, 100 and 1000 µM in the absence and presence of 1 mM salicylate (upper panel) and those from the other neurone in the presence and absence of 10 mM salicylate (lower panel). (B) The concentration–response relationships of the glycine-induced current (I_Gly) in the absence and presence of 1 or 10 mM salicylate. The data were fitted to sigmoidal curve. Note that salicylate depressed the I_Gly without significantly altering the EC₅₀ (48.5 ± 7.6 µM in the absence of salicylate, 42.5 ± 4.3 µM in the presence of 1 mM salicylate and 49.0 ± 8.8 µM in the presence of 10 mM salicylate) and the Hill coefficient (1.48 ± 0.26 in the absence of salicylate, 1.54 ± 0.19 in the presence of 1 mM salicylate and 1.40 ± 0.28 in the presence of 10 mM salicylate) (P > 0.05, one-way anova). Also note that salicylate effectively inhibited the maximal current induced by glycine at a high concentration (3 mM) (P < 0.05, one-way anova). Arrowhead indicates that all the data are normalized to the current induced by 100 µM glycine. Each point represents the averaged value from 5 to 14 neurones. Vertical bars represent ±SEM.

Figure 2

Intracellular salicylate (NaSal) dialysis had no effects on the inhibition of the I_Gly by salicylate. (A) Sample recordings of the I_Gly with a micropipette containing 1 mM salicylate under conditions a (before the dialysis), b (following intracellular salicylate dialysis for 15 min) and c (in the presence of extracellular 1 mM salicylate following the dialysis). (B) Summary data for the I_Gly recorded from five neurones under conditions a, b and c. Note the I_Gly was not changed by the dialysis (condition b) but changed by extracellular application of salicylate following the dialysis (condition c). The I_Gly is normalized to that recorded under condition a (before the dialysis and in the absence of salicylate). Vertical bars represent SEM. NS indicates no significant difference. ** indicates P < 0.01.

Figure 3

Salicylate (NaSal) depressed the I_Gly only when applied simultaneously with glycine (Gly). (A) Sample traces of the I_Gly recorded with three different drug application protocols. In protocol a, the neurone was pretreated with salicylate for 15 s and then exposed to salicylate and glycine simultaneously; in protocol b, the neurone was exposed to glycine alone immediately after 15-s pretreatment of salicylate; in protocol c, the neurone was exposed to salicylate and glycine simultaneously without pretreatment of any drugs. (B) Summary data for the I_Gly recorded from six neurones with the three application protocols. Note that salicylate depressed the I_Gly only when applied with glycine simultaneously (with protocols a and c). The I_Gly is normalized to the response induced by 100 µM glycine alone. Vertical bars represent SEM. NS indicates no significant difference. ** indicates P < 0.01.

Figure 4

Salicylate (NaSal)-induced decrease in glycine-activated membrane conductance was voltage-dependent. (A) Voltage-ramp protocol used to derive the current–voltage relationship. In this protocol, the first ramp occurred in the absence of drug and the second ramp in the presence of drug. Current–voltage relationship was derived by subtracting the current trace produced by the first ramp from that produced by the second ramp. (B) Example of derived current–voltage relationship for a neurone in the absence and presence of 1 mM salicylate. The relationship was corrected for junction potentials. The reversal potential in this example was −2.89 mV and −3.60 mV in the absence and the presence of 1 mM salicylate respectively. Inset shows the averaged reversal potentials (E_reversal) from five neurones. NS indicates no significant difference. (C) Summary data of glycine-activated membrane conductance in the presence of 1 mM salicylate at a negative (−60 mV) and a positive (+60 mV) holding potentials (n= 9). Note that salicylate decreased the conductance to a larger extent at the positive potentials than at the negative potentials. The conductance is normalized to the response in the absence of salicylate. Vertical bars represent SEM. ** indicates P < 0.01.

Figure 5

Salicylate (NaSal) specifically inhibited the current mediated by glycine receptors containing α1-subunits expressed in HEK293T cells. (A) Sample traces of the current mediated by different recombinant glycine receptors in the absence and presence of 1 mM salicylate. (B) Summary data showing the effects of 1 mM salicylate on the I_Gly. Note that salicylate only inhibited the current mediated by recombinant α1- or α1β-glycine receptors. The I_Gly is normalized to the response in the absence of salicylate. Sample sizes are indicated in parentheses. Vertical bars represent SEM. NS indicates no significant difference. * indicates P < 0.05.

Figure 6

A single site mutation of isoleucine to valine at position 240 in the α1-subunit abolished action of salicylate on α1-glycine receptors expressed in HEK293T cells. (A) Aligned sequences showing that near the pore-forming region, the α1-subunit is different from the α2-/α3-subunit only in residues at positions 240 in TM1 and 254 in TM2. (B) Representative traces (left panel) and summary data (right panel) showing the effects of salicylate (1 and 10 mM) on the currents mediated by α1-glycine receptors of WT, by I240V α1-glycine receptors or by G254A α1-glycine receptors. Note that the mutation I240V, rather than the mutation G254A, abolished action of salicylate on α1-glycine receptors. * indicates P < 0.05 and ** indicates P < 0.01. (C) Representative traces (left panel) of the currents mediated by WT α1-glycine receptors and by I240V α1-glycine receptors. Summary data (right panel) for the concentration–response relationships of the currents mediated by WT ?α1-glycine receptors and by I240V α1-glycine receptors. Data are normalized to the maximal response. Note that the EC₅₀ values for mutant I240V α1-glycine receptors and for WT α1-glycine receptors were not significantly different (34.3 ± 5.5 µM vs. 30.0 ± 5.3 µM) (P > 0.05, one-way anova). Sample sizes are indicated in parentheses. TM1, first transmembrane segment; TM2, second transmembrane segment; WT, wild type.

Figure 7

A single site mutation of V247I in the α2-glycine receptors and V240I in α3-glycine receptors made the receptor become sensitive to salicylate. (A) Representative traces (left panel) and summary data (right panel) showing the effects of salicylate (1 and 10 mM) on the currents mediated by wild-type (WT) α2-glycine receptors and V247I α2-glycine receptors. (B) Representative traces (left panel) and summary data (right panel) showing the effects of salicylate (1 and 10 mM) on the currents mediated by WT α3-glycine receptors and V240I α3-glycine receptors. The current is induced by 100 µM glycine and normalized to the response in the absence of salicylate (dashed lines). Sample sizes are indicated in parentheses. Vertical bars represent SEM. ** indicates P < 0.01 and *** indicates P < 0.001.