A novel, rapid, inhibitory effect of insulin on alpha1beta2gamma2s gamma-aminobutyric acid type A receptors

Abstract

In the CNS, GABA and insulin seem to contribute to similar processes, including neuronal survival; learning and reward; and energy balance and food intake. It is likely then that insulin and GABA may interact, perhaps at the GABA(A) receptor. One such interaction has already been described [Q. Wan, Z.G. Xiong, H.Y. Man, C.A. Ackerley, J. Braunton, W.Y. Lu, L.E. Becker, J.F. MacDonald, Y.T. Wang, Recruitment of functional GABA(A) receptors to postsynaptic domains by insulin, Nature 388 (1997) 686-690]; in it a micromolar concentration of insulin causes the insertion of GABA(A) receptors into the cell membrane, increasing GABA current. I have discovered another effect of insulin on GABA(A) currents. Using a receptor isoform, alpha(1)beta(2)gamma(2s) that is the likely main neuronal GABA(A) isoform expressed recombinantly in Xenopus oocytes, insulin inhibits GABA-induced current when applied simultaneously with low concentrations of GABA. Insulin will significantly inhibit currents induced by EC(30-50) concentrations of GABA by about 38%. Insulin is potent in this effect; IC(50) of insulin was found to be about 4.3 x 10(-10) M. The insulin effect on the GABA dose responses looked like that of an antagonist similar to bicuculline or beta-carbolines. However, an effect of phosphorylation on the GABA(A) receptor from the insulin receptor signal transduction pathway cannot yet be dismissed.

Figure 1. Effect of insulin on GABA-mediated currents at α₁β₂γ_2S receptors

All currents are from α₁β₂γ_2s GABA_A receptors expressed in Xenopus oocytes clamped at −60 mV. A. The first series of tracings shows that 100 nM insulin inhibits currents elicited by 1 µM GABA. The first two tracings are controls; the bar corresponds to the application of 1 µM GABA only (G). The next two are tracings of 1 µM GABA co-applied with 100 nM insulin with the bar corresponding to the time of co-application (G + I). B. The second series of tracings shows that 1 nM insulin inhibits currents elicited by 1 µM GABA. The first two tracings are the controls; the bar corresponds to the application of 1 µM GABA only (G). The third and fourth tracings are the currents of GABA co-applied with 1 nM insulin with the bar corresponding to the time of co-application (G + I). The last trace shows that the inhibitory effect of insulin is reversible with the bar corresponding to the application of 1 µM GABA only (G). C. The third series of tracings shows no significant effect of 100 nM insulin in the presence of 100 µM GABA. The first two tracings the controls; the bar corresponds to the application of 1 µM GABA only (G). The last two tracings are the GABA currents in the presence of insulin with the bar corresponding to the time of co-application (G + I).

Figure 2. Insulin dose response at 1 µM GABA for α₁β₂γ_2s receptors

Various concentrations of insulin (100 nM to 0.01 nM) were added with 1 µM GABA. The percent effect is the change in current from control currents (no insulin). Points are the average and standard deviations of 3–6 experiments. The curve was fit using a two site model on GraphPad. Insulin effects on currents are statistically significant at 100 nM and 10 nM (p <0.05). The maximal effect of insulin is approximately −38%, with an IC₅₀ in the 4.3 × 10⁻¹⁰ range.

Figure 3. GABA current changes in the presence of 100 nM insulin at α₁β₂γ_2s receptors

The change in GABA-induced current due to the presence of 100 nM insulin is plotted as a function of GABA concentration. GABA EC₅₀ is approximately 4 µM and shifts to 15 µM in the presence of insulin. Points are mean and standard deviations of 3–5 experiments.