Rapid upregulation of alpha7 nicotinic acetylcholine receptors by tyrosine dephosphorylation

Abstract

Alpha7 nicotinic acetylcholine receptors (nAChRs) modulate network activity in the CNS. Thus, functional regulation of alpha7 nAChRs could influence the flow of information through various brain nuclei. It is hypothesized here that these receptors are amenable to modulation by tyrosine phosphorylation. In both Xenopus oocytes and rat hippocampal interneurons, brief exposure to a broad-spectrum protein tyrosine kinase inhibitor, genistein, specifically and reversibly potentiated alpha7 nAChR-mediated responses, whereas a protein tyrosine phosphatase inhibitor, pervanadate, caused depression. Potentiation was associated with an increased expression of surface alpha7 subunits and was not accompanied by detectable changes in receptor open probability, implying that the increased function results from an increased number of alpha7 nAChRs. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis was shown to be a plausible mechanism for the rapid delivery of additional alpha7 nAChRs to the plasma membrane. Direct phosphorylation/dephosphorylation of alpha7 subunits was unlikely because mutation of all three cytoplasmic tyrosine residues did not prevent the genistein-mediated facilitation. Overall, these data are consistent with the hypothesis that the number of functional cell surface alpha7 nAChRs is controlled indirectly via processes involving tyrosine phosphorylation.

Figure 5.

A, Concentration-response curves for ACh before and after treatment with 10 μm genistein. All data points were normalized to the response induced by 100 μm ACh alone. B, Representative traces showing that genistein-mediated potentiation was present over a wide range of ACh concentrations (30-3000 μm). Responses at concentrations of 30 and 300 μm ACh were from the same oocyte, whereas those at 3 mm ACh were from a different cell.

Figure 1.

α7 nAChR function is modulated by inhibitors of tyrosine kinases and tyrosine phosphatases. A, Representative examples of ACh-induced α7 currents before, during, and after 5 min applications of 10 μm genistein (top traces) and 10 μm pervanadate (bottom traces). B, Plot showing the concentration dependence of potentiation of currents by genistein. C, Summary histogram illustrating the mean effects of inhibition or activation of PTKs and PTPs on α7 currents. The concentrations of drugs were as follows: genistein, 10 μm; pervanadate, 10 μm; insulin, 5 μm; daidzein, 10 μm; PP2, 10 μm.

Figure 2.

Potentiation is rapid, reversible, and does not result from a direct interaction of genistein with α7 nAChRs. A, Plots showing the time courses of the onset of and recovery from the effects of genistein. Solid lines represent single exponents fitted to the data. τ_on and τ_off are the exponential time constants for onset and recovery, respectively. B, Plot illustrating (open symbols) the lack of effect of coapplication of 10 μm genistein (filled bars) and ACh (open bars) on the peak amplitude of α7 receptor-mediated currents. Applications were at 5 min intervals. Inset, Example traces of ACh-induced responses in the absence and presence of genistein.

Figure 3.

The action of genistein is specific to α7 nAChRs. The effects of genistein (10 μm) treatment on ACh-induced currents in oocytes expressing either α4β2 (A) or α3β4 (B) receptors are shown. C, Histogram of the mean effects of genistein on these two classes of receptors.

Figure 4.

Native α7 nAChRs are regulated by tyrosine kinases and phosphatases. A, Example traces showing responses of hippocampal stratum radiatum interneurons to local pressure application of choline (10 mm), before and during administration of 10 μm genistein (top) or 10 μm pervanadate. B, Time course of the effects of genistein and pervanadate on choline-induced peak currents. Drugs were administered continuously via the bath starting at t = 0 min. In the control condition (open symbols), slices were superfused with control ACSF only. C, Histogram illustrating the reversibility of a 5 min application of genistein in a separate series of experiments. The holding potential of neurons was -70 mV.

Figure 6.

Surface expression of α7 receptors is increased by genistein. A, α7 inmmunoreactivity in oocytes. Data are from 10 oocytes per lane. Immunoblotting conditions for each lane are listed below the gel. B, The effects of genistein on nAChR surface expression.α7-expressing oocytes were left untreated or treated for 5 min before surface biotinylation with 10 μm genistein. Representative immunoblot shows α7 immunoreactivity in surface biotinylated (B) and nonbiotinylated (NB) fractions. Data from three such experiments for biotinylated (filled bars) and nonbiotinylated (open bars) fractions are quantified in the graph from densitometry measurements and plotted relative to total α7 immunoreactivity. Experimental conditions that resulted in a significant change (p < 0.05) from control values are denoted by the asterisk. C, Specific binding of αBTX to the surface of intact oocytes (n = 5 per concentration) in the absence (open symbols) and presence (filled symbols) of 10 μm genistein. Specific binding was obtained by subtracting background during competition with either ACh (300 μm) or choline (10 mm). D, Specific αBTX binding to hippocampal cells showing both surface and total binding for each of three conditions: control, genistein (gen; 10 μm), daidzein (daid; 10 μm).

Figure 7.

The open probability of α7 nAChRs is not affected by tyrosine kinase inhibition. A, Examples showing the inhibition of α7 responses during coapplication of ACh (30 μm) and chlorisondamine (5 μm) in the absence (top traces) or continuous presence (bottom traces) of 10 μm genistein. The mean ratio of the ACh-induced response immediately after coapplication compared with the response before coapplication with chlorisondamine (blocking index) is plotted as a function of the ACh concentration in the histogram (B). C, Test of the usefulness of chlorisondamine to detect changes in open probability. The difference in the blocking index resulting from coapplication of both 30 and 300 μm ACh and 5 μm chlorisondamine is plotted as a histogram.

Figure 8.

Endocytosis is not involved in upregulation of α7 receptors. A, Representative currents induced by application of a cAMP agonist mixture for 2 min in oocytes coexpressing CFTR with either wild-type (wt) or K44A dynamin. The holding potential was -50 mV. B, Histogram of the mean CFTR-mediated currents induced by the cAMP agonist mixture. C, Histogram of the mean currents induced by ACh (100 μm) in oocytes coexpressing α7 nAChRs with either wild-type or K44A dynamin. The holding potential was -65 mV. D, Histogram showing the effect of genistein (10 μm) on α7 receptor-mediated responses in oocytes expressing either wild-type or K44A dynamin. E, Representative examples of the genistein-mediated potentiation of α7 currents after MβC pretreatment (10 μm; 60-90 min). F, Histogram illustrating the effects of MβC pretreatment.

Figure 9.

SNARE-mediated exocytosis is involved in the augmentation of α7 nAChRs by genistein. A, Examples of currents induced by the coagonists NMDA (300 μm) and glycine (10 μm) in oocytes expressing NR1a and NR2b subunits before and after exposure to insulin (5 μm; 10 min) 5-7 h after injection of vehicle (control; left two traces) or with 50 ng of BoNT-A (right two traces). B, Histogram illustrating the effects of BoNT-A on the insulin-mediated potentiation of NMDA receptors. C, Examples of currents induced by ACh (100 μm) in oocytes expressing α7 subunits before and after exposure to genistein (10 μm; 5 min) 5-7 h after injection of vehicle (control; left two traces) or with 1 ng of BoNT-A (right two traces). D, Histogram illustrating the effects of BoNT-A on the genistein-mediated potentiation of α7 nAChRs.

Figure 10.

The actin cytoskeleton is not involved in the genistein-induced regulation of α7 nAChRs. A, Histogram showing the mean effects of cyt D pretreament on genistein-induced potentiation of α7 receptor-mediated responses. B, Examples traces of α7 receptor-mediated currents induced by ACh (100 μm) in control medium 5-10 min after cyt D (10 μm) treatment, 5 min after coapplication of cyt D and genistein (10 μm), and after wash.

Figure 11.

The effects of genistein do not involve tyrosine residues on α7 subunits. A histogram comparing the potentiation by genistein (10 μm) of mutant α7 subunits to wild-type α7 subunits is shown. Mutant subunits contained either a single point mutation at each of the three tyrosine residues (Y317, Y386, and Y442) or a triple mutation of all three residues (triple mutant). Each subunit, mutants and wild type, were expressed separately in oocytes and tested after a 5 min incubation with genistein.