Low concentrations of nicotine differentially desensitize nicotinic acetylcholine receptors that include α5 or α6 subunits and that mediate synaptosomal neurotransmitter release
Highlights
► Desensitization of presynaptic nAChRs was assessed by neurotransmitter release. ► α4α5β2-nAChR recovers from desensitization more quickly than α4β2-nAChR. ► The desensitization concentration for α4α5β2-nAChR is higher than for α4β2-nAChR. ► The extent of desensitization of α6β3β2-nAChR is less than α4β2- or α4α5β2-nAChR.
Introduction
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels known to desensitize with prolonged exposure to agonists (Changeux et al., 1984). A cyclical scheme to explain the process of desensitization of nAChRs at the neuromuscular junction was proposed in 1957 by Katz and Thesleff (Katz and Thesleff, 1957). Since that time it has been recognized that all nAChRs are allosteric proteins that desensitize with varying kinetics (Changeux et al., 1984; Quick and Lester, 2002; Taly et al., 2009). Multiple states of desensitization and/or inactivation exist and many factors, including subunit composition, association with other proteins, lipids, ions, and/or accessory molecules (possibly unique to each cell-type), as well as post-translational modifications, are likely to affect desensitization and inactivation (Ibanez-Tallon et al., 2002; Cheng et al., 2009; Neff et al., 2009; Taly et al., 2009).
The β2∗-subtype is the most abundant nAChR in the CNS [∗ indicates presence of other subunits (Lukas et al., 1999)]. The propensity of the β2∗-nAChR to desensitize with long-term exposure to even low concentrations of agonists, such as nicotine, is a complex property of these receptors (Lippiello et al., 1987; Fenster et al., 1999; Quick and Lester, 2002; Gentry et al., 2003; Guo and Lester, 2007; Taly et al., 2009; Papke et al., 2011). Desensitization by an agonist depends on the concentration and length of exposure, with long exposures resulting in deeper states of desensitization that recover more slowly (Quick and Lester, 2002).
The most common β2∗-nAChR is the α4β2-nAChR which exists in both a high sensitivity (HS) form with the subunit stoichiometry (α4β2)2β2 and a low sensitivity (LS) form (α4β2)2α4 (Zwart and Vijverberg, 1998; Zhou et al., 2003; Marks et al., 2007; Gotti et al., 2008). The diversity of α4β2∗-nAChR is further complicated by the α5 subunit, expressed in certain neurons that can assemble as (α4β2)2α5-nAChR, another HS form (Tapia et al., 2007; Bailey et al., 2010). The α5 subunit is an accessory subunit and not part of the ligand binding domain (Kuryatov et al., 2008), although recently a ligand binding site at the α4α4 interface has been described (Harpsoe et al., 2011; Mazzaferro et al., 2011), raising the possibility that α4α5 or α5β2 may be tertiary binding sites. The role of the α5 subunit in altering function of α4β2∗-nAChRs has been studied in expression systems. In one study in oocytes, addition of the α5 subunit resulted in larger currents and more desensitization (Ramirez-Latorre et al., 1996). In another study using tandem constructs to better control populations of receptors, the (α4β2)2α5-nAChR showed equally high sensitivity as (α4β2)2β2, but with considerably increased Ca++ permeability (Tapia et al., 2007). The HS nature of the (α4β2)2α5-nAChR has also been demonstrated in mouse brain by 86Rb+ efflux experiments using synaptosomal preparations from striatum and thalamus (Brown et al., 2007) and in a transgenic mouse in cortico-thalamic terminals (Heath et al., 2010), as well as by [3H]-GABA release in cortex (McClure-Begley et al., 2009) and [3H]-DA release in striatum (Grady et al., 2010a). In layer VI cortical neurons, the α5 accessory subunit forms HS receptors with α4 and β2 subunits, and the presence of the α5 subunit protects against desensitization (Bailey et al., 2010).
Another important but less widely expressed β2∗-nAChR contains the α6 subunit as a ligand binding subunit, and forms nAChRs of predominately the (α6β2)2β3 and (α6β2)(α4β2)β3 subtypes which are expressed in DAergic terminals [for review see (Quik et al., 2011)]. Both of these subtypes are HS forms, with the latter having the highest sensitivity for activation by nicotine of any reported subtype (Salminen et al., 2007).
All HS subtypes respond to low concentrations of nicotine [EC50 values of ∼1 μM or lower and nM DC50 values (half-maximal effective concentration for desensitization)]. These subtypes will mediate effects of nicotine in vivo [with concentrations measured in venous blood of smokers reported to be in the range of 10–50 ng/ml equivalent to 60–310 nM (Jarvik et al., 2000; Brody, 2006).
Furthermore, genome wide association studies have implicated the α5 and α6 subunits in aspects of smoking dependence [for review see (Greenbaum and Lerer, 2009)]. Thus, information on how these subunits influence desensitization properties of the β2∗-nAChR is of major importance in understanding the actions of nicotine as well as the actions of various therapeutic drugs (Picciotto et al., 2008; Andreasen et al., 2009; Buccafusco et al., 2009; Mansvelder et al., 2009; Taly et al., 2009).
This study was undertaken to assess the roles of α5 and α6 subunits on desensitization properties of β2∗-nAChRs from native tissues which have intact the accessory molecules and post-translational modifications unique to neurons. Using α5 subunit null mutant mice (α5KO) as well as wild-type (WT) mice, desensitization of (α4β2)2β2 and (α4β2)2α5 was studied on GABA terminals. Similar methods with α5KO and α6KO mice and the selective antagonist α-conotoxin MII (α-CtxMII) were used to compare these same subtypes on DAergic terminals. In addition, using α4KO mice, the desensitization of α6β2∗-nAChR on dopaminergic terminals was assessed.
Section snippets
Mice
Mice were bred and housed at the Institute for Behavioral Genetics, University of Colorado (Boulder, CO). All subunit null mutant mice were bred onto the C57BL/6 background for minimum of 10 generations. A 12 h light/dark cycle was maintained (lights on 7AM to 7PM), at 22 °C and mice had free access to food (Teklad Rodent Diet, Harlan, Madison WI) and water. Genotypes were assessed by PCR as previously described (Salminen et al., 2004, 2007). The α4 null mutant mice (α4KO) were originally
Measurement of desensitization
Neurotransmitter release assays measure an event that occurs several steps beyond the actual ion flux through the nAChR. Consequently, the potential for measuring desensitization of other steps in the chain of events leading to neurotransmitter release exists. However, in order to measure the effect of the α5 and α6 subunits, this approach may be our only way of isolating populations of nAChR. Therefore, we explored the use of Ca++-free buffer during the desensitizing exposure to see if we
α4β2- and α4α5β2-nAChR
Experiments presented here utilized relatively short term (10 min) exposures to a nicotine concentration (300 nM) likely to be found in plasma of tobacco smokers (Matta et al., 2007) to study the desensitization of nAChRs mediating neurotransmitter release from mouse brain synaptosomes. The exposure to nicotine was conducted in the absence of external Ca++ to eliminate or minimize any Ca++-induced changes elicited by nicotine and any subsequent down-stream events. Under these conditions,
Acknowledgments
This work was supported by NIH grants: U19DA019375 and P30DA015663.
We thank J. Michael McIntosh for generously supplying α-conotoxin MII, William Van Morter for animal production and care, and Erin Meyers and Esteban Loetz for genotyping.
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Present address: Molecular and Cellular Pharmacology Program, Department of Neuroscience University of Wisconsin, Madison, WI 53706, United States.