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  • Ethanol decreased the value of desensitization for GluR Di

    2021-09-17

    Ethanol decreased the τ-value of desensitization for GluR-Di. Coexpression of stargazin or γ4 further increased the effect of ethanol on desensitization (Fig. 3A, B). The highest ethanol concentration tested (200mM) reduced the τ-value by 16.5±4.9% (n=5) in the control receptors and by 29.3±9.3% (n=6) in the stargazin coexpressed receptors. Ethanol (200mM) lowered the τ-value by 14.4±5.8% (n=5) and 34.2±5.0% (n=7) with and without γ4 coexpression, respectively. We also studied the recovery from desensitization by applying two 200-ms pulses of 10-mM glutamate to the GSK1363089 with variable intervals ranging from 25 to 600ms. With this application protocol, the current evoked by the second pulse had a smaller amplitude as compared with that produced by the first one with short interpulse intervals, before it was fully recovered from desensitization (Fig. 4A). The peak current amplitude of the second pulse was divided by that of the first one giving the P2/P1 ratio. The ratios were plotted against the pulse intervals, and the points fitted to a single exponential equation, giving then τrec-values, the time constants of recovery from desensitization. The recovery from desensitization for the GluR-Di was not altered by the coexpression of stargazin, but γ4 significantly prolonged it (Fig. 4B). The τrec-values were 31ms (95% confidence interval: 27–37ms, n=7), 31ms (29–33ms, n=7), and 59ms (53–66ms, n=5) for GluR-Di alone, GluR-Di+stargazin, and GluR-Di+γ4, respectively. In contrast to our previous observation in isolated hippocampal neurons (Möykkynen et al., 2003), ethanol did not affect recovery from desensitization (Fig. 4C–E). Ethanol inhibition is markedly decreased in the nondesensitizing point-mutated L497Y GluR-Ai receptors expressed in HEK-293 cells (Möykkynen et al., 2003). We wanted to test whether ethanol inhibition is also decreased with the analogous point mutation constructed in GluR-Di receptors. Application of 10mM glutamate evoked a current that had a small but distinct peak current (approximately 9% higher than GSK1363089 steady-state current) indicating that the L505Y GluR-Di receptors still undergo desensitization to some extent. Ethanol inhibited both the peak and steady-state currents of L505Y GluR-Di equally, and, therefore, only the steady-state current was used in further analysis. The steady-state current of L505Y GluR-Di receptors was significantly less inhibited by ethanol than the steady-state current of wild type GluR-Di receptors (200mM ethanol inhibited L505Y only by 23.9±7.1%, n=6 and wild type by 58.1±7.2%, n=6) (Fig. 5B).
    Discussion In this study, ethanol inhibited the glutamate-evoked currents of GluR-Di receptors to the same extent as in previous studies the currents mediated by native AMPA receptors (Möykkynen et al., 2003, Wirkner et al., 2000). The steady-state current was inhibited more than the peak current for all GluR-D receptors. The coexpression with TARPs did not affect the differential ethanol sensitivity of the peak and steady-state current components. Interestingly, we observed that ethanol accelerated the onset of desensitization, revealed as shortening of the τ-value of desensitization, and this effect was further enhanced in receptors cotransfected with TARPs. This finding provides direct evidence for the idea that ethanol increases desensitization of AMPA receptors. Previously, increased desensitization was suggested to play a role in ethanol inhibition on the basis of indirect evidence—blocking the desensitization by cyclothiazide and using nondesensitizing point-mutated GluR-Ai receptors expressed in HEK-293 cells reduced the ethanol inhibition (Möykkynen et al., 2003). Important molecular domains for AMPA receptor opening and desensitization are the ligand-binding domain and a linker domain between the ligand-binding domain and the channel pore (Armstrong and Gouaux, 2000, Armstrong et al., 2006, Horning and Mayer, 2004, Sun et al., 2002). The ligand-binding domain forms a clamshell-like binding cleft as a combination of two extracellular S1 and S2 domains of AMPA receptors (Stern-Bach et al., 1994). When an agonist binds to the binding cleft, the cleft is thought to close, which stretches the linker domain causing pressure on the channel pore area. The pressure then conveys to channel opening or desensitization. TARPs make physical contact with the AMPA receptor ligand-binding domain at the first extracellular loop (Tomita et al., 2007). At least two different mechanisms for TARP effects on AMPA receptor gating have been suggested (reviewed in Milstein and Nicoll, 2008). TARPs may increase the binding cleft closure or they might interact directly with the linker domain. Both of these mechanisms could increase the pressure on the channel pore region mediated by the linker domain. It is not known how ethanol interacts with AMPA receptors at the molecular level. There are, however, data suggesting that ethanol has a binding pocket in AMPA receptors. Alcohols exhibit a cutoff phenomenon for AMPA receptor inhibition. The potency of n-alcohols to inhibit AMPA receptors function grows with increasing carbon chain length, but only up to heptanol (Akinshola, 2001). This most likely means that ethanol has a binding pocket within a receptor domain that is accessible via a hydrophobic milieu, such as lipid cell membrane. Ethanol binding pocket may, therefore, be located in close proximity to the pore-forming regions of the AMPA receptors. Regarding desensitization of the AMPA receptor, ethanol and TARPs have opposite effects—TARPs slow and decrease desensitization, whereas ethanol speeds up and increases it. Ethanol may, therefore, influence the channel pore region hindering the opening of the channel and favoring the desensitized state.