Cross Talk between α7 and α3β4 Nicotinic Receptors Prevents Their Desensitization in Human Chromaffin Cells

Abstract

The physical interaction and functional cross talk among the different subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in the various tissues is unknown. Here, we have investigated this issue between the only two nAChRs subtypes expressed, the a7 and a3b4 subtypes, in a human native neuroendocrine cell (the chromaffin cell) using electrophysiological patch-clamp, fluorescence, and Förster resonance energy transfer (FRET) techniques. Our data show that a7 and a3b4 receptor subtypes require their mutual and maximal efficacy of activation to increase their expression, to avoid their desensitization, and therefore, to increase their activity. In this way, after repetitive stimulation with acetylcholine (ACh), a7 and a3b4 receptor subtypes do not desensitize, but they do with choline. The nicotinic current increase associated with the a3b4 subtype is dependent on Ca 21 . In addition, both receptor subtypes physically interact. Interaction and expression of both subtypes are reversibly reduced by tyrosine and serine/threonine phosphatases inhibition, not by Ca 21 . In addition, expression is greater in human chromaffin cells from men compared to women, but FRET efficiency is not affected. Together, our findings indicate that human a7 and a3b4 subtypes mutually modulate their expression and activity, providing a promising line of research to pharmacologically regulate their activity.

α3β4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells

“…In the latter case, nAChRs would already be at the vicinity of the exocytotic active sites, so that a faster and/or more efficient response would be achieved. Recently, it has been shown in human chromaffin cells that repeated ACh stimulation potentiates nAChR currents and prevents nAChR desensitization, in a process that depends on α3β4 and α7 nAChRs [ 30 ]. It seems evident that this potentiation, together with the increased proximity between α3β4 nAChR clusters and the active sites of exocytosis that we observed, could result in a further enhancement of the secretory process.…”

“…Chromaffin cells were isolated from bovine adrenal glands by collagenase digestion and they were further separated from the debris by centrifugation on Percoll gradients. After washing, they were resuspended with Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Madrid D-5796, stored 4 °C) to obtain a cell suspension, as described elsewhere [ 30 ].…”

Nicotinic Receptors in Human Chromaffin Cells: Characterization, Functional and Physical Interactions between Subtypes and Regulation

“…Later on, we described the presence of functional α7-nAChRs using electrophysiological techniques and α7-nAChRs antagonists such as α-BgTx (1 µM) and MLA (10 µM) [61]. It is very relevant to mention that the expression of functional α7-nAChRs increases with repeated stimulation with ACh or another agonist that completely and simultaneously activates the α7and α3β4*-nAChRs ( [62], see below). In addition, an almost full block of the β4*-nAChRs currents and exocytosis (99.6% and 94%, respectively) was achieved in these cells using the α-Ctx BuIA (T5A,P60) (BuIA, selective for β4*-nAChRs; [63]) [64].…”

“…We found it to be of interest to investigate it in a human cell that express mainly two of these receptors, making it easier to find out the mechanism of interaction between them and its consequences. We designed the following experiments to investigate it [62].…”

Adaptation of the adrenal medulla to stress: Pathophysiological implications

“…En todas las especies animales estudiadas (hombre, vaca, rata y ratón) se ha descrito la presencia de las subunidades 3 y 7, capaces de formar receptores homoméricos (7) y heteroméricos (incorporan otras subunidades como la 2, 3, 4, 5, 6, b2 y b4) (15,16). En las células cromafines humanas se ha descrito que la interacción física entre los receptores 7 y los 3b4 permite prevenir su desensibilización cuando los dos receptores son activados simultáneamente (17). El hecho de que la expresión del receptor potenciales de acción (8,9).…”