Elsevier

Cell Calcium

Volume 39, Issue 4, April 2006, Pages 349-355
Cell Calcium

Ca2+ permeability through rat cloned α9-containing nicotinic acetylcholine receptors

https://doi.org/10.1016/j.ceca.2005.12.002Get rights and content

Abstract

We investigated the functional properties of rat α9 and α9α10 nicotinic acetylcholine receptors (nAChRs) expressed by transient transfection in the rat GH4C1 cell line, using both Ca2+ imaging and whole-cell recording. Acute applications of ACh generated short-delay fast-rising and quick-decaying Ca2+ transients, suppressed in Ca2+-free medium and invariably accompanied by the activation of whole-cell inward currents. The mean amplitude of ACh-induced currents was as small as −16 pA in α9 subunit cDNA-transfected GH4C1 cells (α9-GH4C1), while they were much larger (range: −150 to −300 pA) in α9α10 subunit cDNAs-transfected GH4C1 cells (α9α10-GH4C1). Currents were not activated by nicotine, were blocked by methyllycaconitine and were ACh concentration-dependent. Because the Ca2+ permeability of α9-containing nAChRs has been estimated in immortalized cochlear UB/OC-2 mouse cells, we also characterized the ACh-induced responses in these cells. Unlike α9- and α9α10-GH4C1 cells, UB/OC-2 cells responded to ACh with both long-delay methyllycaconitine-insensitive whole-cell currents and long-lasting Ca2+ transients, the latter being detected in the absence of Ca2+ in the extracellular medium and being suppressed by the Ca2+-ATPase inhibitor thapsigargin, known to deplete IP3-sensitive stores. These results indicated the involvement of muscarinic nAChRs and the lack of functional ACh-gated receptor channels in UB/OC-2 cells. Thus, we measured the fractional Ca2+ current (Pf, i.e. the percentage of total current carried by Ca2+ ions) in α9α10-GH4C1, obtaining a Pf value of 22 ± 4%; this is the largest value estimated to date for a ligand-gated receptor channel. The physiological role played by Ca2+ entry through α9-containing nAChRs gated by ACh is discussed.

Introduction

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric proteins formed by the combination of α2-α10 and β2-β4 subunits, gating cationic channels and playing a key role in some neurological disorders [1], [2], [3], [4], [5]. Because, in addition to mediating fast neurotransmission, the functional role of nAChRs is related to their Ca2+ permeability [6], in the last few years there has been a great deal of interest in measuring the Ca2+ permeability of nAChRs [7].

Ca2+ permeates the receptor pore to various extents [7], and its permeability is determined by the subunit composition, being relatively high when the nAChRs are formed by α7-α10 subunits [8], [9], [10], [11], [12], [13], [14] and relatively low when the nAChRs are formed by α2-α6 together with β2-β4 subunits [15], [16], [17]. Two experimental approaches are currently used to determine the Ca2+ entry through the nAChR-channel pore: the shift in the ACh current reversal potential as a function of external Ca2+ and the simultaneous recording of both Ca2+ transients by fluorescence microscopy and transmembrane currents by patch-clamp techniques [15], [18]. The latter approach is a direct method (used in this work) yielding to the determination of the fractional Ca2+ current (Pf), which is the percentage of total ACh-induced current carried by Ca2+ ions.

The α9-containing nAChRs are expressed in several cell systems including: (i) cochlear outer hair cells [19]; (ii) dorsal root ganglia [20], [21]; (iii) keratinocytes [22]; (iv) lymphocytes [23], [24]; and (v) human sperm [25]. Thus, given this heterogeneous distribution of α9-containing nAChRs, any information about its Ca2+ permeability may be physiologically relevant. While the Pf values of many nAChRs have been determined in native or heterologous cell systems [7], the Pf values of α9-containing nAChRs are lacking. A large Ca2+ permeability has been suggested for both α9- and α9α10-nAChRs, with PCa/PNa values ranging from 8 to 16 reported elsewhere [9], [14], [26], [27]. In this work we performed experiments to investigate the properties of Ca2+ transients elicited by the activation of α9-containing nAChRs and to determine their Pf value when transiently expressed in GH4C1 cells.

Section snippets

Expression of both α9- and α9α10-nAChRs in rat GH4C1 cells

The neuronal rat α9 and α9α10 subunits, kindly provided by Dr. J. Boulter, were expressed by transient transfection in the rat anterior pituitary GH4C1 cells, plated at a density of 5–10 × 104 in 35-mm Petri dishes, and grown in HAM F10 nutrient mixture, with 10% foetal bovine serum, 1% penicillin and streptomycin. Transient transfection was achieved by adding to each dish 1 μg of α9 subunit cDNA (together with 1 μg of α10 subunit cDNA, when necessary), along with 3 μl of Fugene (Roche,

Activation of α9-containing nAChRs generates ACh-evoked Ca2+ transients

The application of ACh (100 μM, 2 s duration) evoked Ca2+ transients with ΔR = 1.5 ± 0.2 in 14 out of the total 129 examined GH4C1 cells transfected with α9 subunit cDNA (α9-GH4C1; ∼11% of transfection efficiency; Fig. 1A); and with ΔR = 2.4 ± 0.9 in 33 out of the total 154 tested cells co-transfected with both α9 and α10 subunit cDNAs (α9α10-GH4C1; ∼21% of transfection efficiency; Fig. 1B). No ACh-induced Ca2+ transients were evoked in cells transfected with α10 subunit cDNA or in non-transfected GH4C1

Discussion

The main goal of this study was to investigate the Ca2+ permeability of rat α9-containing nAChRs expressed in transiently transfected rat pituitary GH4C1 cells. This cell expression system is very useful for predicting the functional properties of cholinergic receptors of native systems because it has already been used to study the Ca2+ permeability of human and rat α7-nAChRs [12], yielding Pf values similar to those measured for the same receptor in native dorsal root ganglion neurons [32].

Acknowledgements

We thank Dr. James Boulter for providing the rat α9- and α10-subunit cDNAs, and Dr. Matthew Holley for providing the UB/OC-2 cell line. We are grateful to Drs. M. Holley and J.F. Ashmore for valuable suggestions and critical reading of the manuscript. This work has been granted by Ministero Università Ricerca (FIRB and PRIN grants to F.E.).

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