Neuronal nicotinic threonine-for-leucine 247 α ₇ mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine

Abstract

Mutation of the highly conserved leucine residue (Leu-247) converts 5-hydroxytryptamine (5HT) from an antagonist into an agonist of neuronal homomeric ␣ 7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We show here that acetylcholine (AcCho) activates two classes of single channels with conductances of 44 pS and 58 pS, similar to those activated by 5HT. However, the mean open time of AcCho-gated ion channels (11 ms) is briefer than that of 5HT-gated ion channels (18 ms). Furthermore, whereas the open time of AcCho channels lengthens with hyperpolarization, that of 5HT channels is decreased. In voltage-clamped oocytes, the apparent affinity of the ␣ 7 mutant receptor for 5HT is not modified by the presence of dihydro-␤-erythroidine, which acts on the AcCho binding site in a competitive manner. This indicates a noncompetitive action of 5HT on nicotinic acetylcholine receptors. Considered together, our findings show that AcCho gates ␣ 7 mutant channels with similar conductance but with different kinetic profile than the channels gated by 5HT, suggesting that the two agonists act on different docking sites. These results will help to understand the crosstalk between cholinergic and serotonergic systems in the central nervous system.

Effects of fluoxetine on wild and mutant neuronal α7 nicotinic receptors

“…12,[24][25][26] Borosilicate-glass patch pipettes (2-4 M⍀ tip resistance) were filled with oocyte Ringer's medium containing ACh (0.2 M) or ACh (0.2 M) plus fluoxetine (10 M). Unitary channel activity was recorded using an Axopatch 200B amplifier (Axon Instruments).…”

“…We have previously shown that oocytes expressing the ␣ 7 mutant receptor exhibit a fairly homogeneous population of ACh-activated channel openings in each oocyte. 12 In five oocytes (two donors) we found that, in the presence of fluoxetine: (i) channels exhibited a marked flickering; (ii) the channel slope conductance was linear but was reduced from 40-72 pS 12 to 20-27 pS; (ii) the open channel duration (range: 5-8 ms at +40 mV pipette potential) became considerably shorter (1-2 ms); and (iii) the frequency of openings, which increased in the controls, 12 was instead markedly reduced by hyperpolarization (Figure 4).…”

Antagonism of nicotinic acetylcholine receptors by inhibitors of monoamine uptake

“…Furthermore, various agonists and antagonists of different 5-HT receptors, as well as inhibitors of the 5-HTtransporter alter the function of neuronal and muscle AChRs. [7][8][9][10][11][12][13][14][15][16][17] The most common action of serotonergic compounds on AChRs appears to be a non-competitive inhibitory process through interactions with the open channel. 5,[7][8][9][10][11][12][13][14] On the other hand, it is known that different classes of inhibitors of monoamine-uptake systems, that are used clinically as antidepressants, interact with different membrane proteins at synaptic regions.…”

The single-channel properties of human acetylcholine α7 receptors are altered by fusing α7 to the green fluorescent protein

“…We constructed a chimera by fusing the enhanced version of GFP to the C terminus region of the human ␣7-AcChoR (␣7-GFP AcChoR) and compared the channel behavior of wild-type (wt) ␣7 vs. ␣7-GFP AcChoRs in Xenopus oocytes. Similar experiments were addressed to the Leu-to-Thr 248 mutant ␣7-AcChoR (mut␣7 AcChoR), which represents a ''gain of function'' model of the homomeric ␣7 receptor because of its unusual pharmacological and functional properties (23)(24)(25)(26)(27). We report that (i) the wt␣7-AcChoR exhibits short channel duration and two-channel conductance levels; (ii) the GFP fusion to the wt␣7-subunit increases the channel duration; and (iii) the GFP fusion to the mut␣7-subunit accelerates channel desensitization.…”