Kinetic properties and open probability of a7 nicotinic acetylcholine receptors Krisztina Pesti a, b , Anett K. Szabo a, b , Arpad Mike b, * , E. Sylvester Vizi c a Semmelweis University, School of Ph.D. Studies, Üll} oi út 26, H-1085 Budapest, Hungary b Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O.B. 67, H-1450 Budapest, Hungary c Laboratory of Drug Research, Institute of Experimental Medicine, Hungarian Academy of Sciences, P.O.B. 67, H-1450 Budapest, Hungary article info Article history: Received 23 March 2013 Received in revised form 13 January 2014 Accepted 21 January 2014 Available online 31 January 2014 Keywords: a7 nicotinic acetylcholine receptor Choline PNU-120596 Kinetic modeling abstract The alpha7 nicotinic acetylcholine receptor (nAChR) has some peculiar kinetic properties. From the literature of a7 nAChR-mediated currents we concluded that experimentally measured kinetic properties reflected properties of the solution exchange system, rather than genuine kinetic properties of the re- ceptors. We also concluded that all experimentally measured EC 50 values for agonists must inherently be inaccurate. The aim of this study was to assess the undistorted kinetic properties of a7 nAChRs, and to construct an improved kinetic model, which can also serve as a basis of modeling the effect of the positive allosteric modulator PNU-120596, as it is described in the accompanying paper. Agonist-evoked currents were recorded from GH4C1 cells stably transfected with pCEP4/rat a7 nAChR using patch-clamp and fast solution exchange. We used two approaches to circumvent the problem of insufficient solution exchange rate: extrapolation and kinetic modeling. First, using different solution exchange rates we recorded evoked currents, and extrapolated their amplitude and kinetics to instantaneous solution ex- change. Second, we constructed a kinetic model that reproduced concentration-dependence and solution exchange rate-dependence of receptors, and then we simulated receptor behavior at experimentally unattainably fast solution exchange. We also determined open probabilities during choline-evoked un- modulated and modulated currents using nonstationary fluctuation analysis. The peak open probability of 10 mM choline-evoked currents was 0.033 Æ 0.006, while in the presence of choline (10 mM) and PNU-120596 (10 mM), it was increased to 0.599 Æ 0.058. Our kinetic model could adequately reproduce low open probability, fast kinetics, fast recovery and solution exchange rate-dependent kinetics. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction The a7 subunit containing nicotinic acetylcholine receptor (a7 nAChR) is known as the second most abundant nicotinic receptor type in the CNS (after the a4b2 type receptor). These receptors mostly but not exclusively are homopentamers of the a7 subunit. Their most prominent role within the central nervous system is probably the pre- and postsynaptic modulation of synaptic function and plasticity (Albuquerque et al., 2009; Alkondon and Albuquerque, 2001; Gray et al., 1996; Gu and Yakel, 2011; Lendvai and Vizi, 2008; Lozada et al., 2012; Rozsa et al., 2008; Vizi and Lendvai, 1999), which is the basis of their role in cognition (Hurst et al., 2012; Lendvai et al., 2012; McKay et al., 2007). The a7 nAChR has some unique properties: a high Ca 2þ permeability, which is counterbalanced by extremely fast desen- sitization, causing a fast onset and decay kinetics and an extremely low open probability (Williams et al., 2012, 2011). The aim of this study was to refine the kinetic analysis of the receptor. In order to clearly see the unusual aspects of a7 nAChR receptor kinetics, we need to briefly review what is known about the kinetic properties of this receptor (Section 3.1 .). This re- examination of reported properties, makes it evident that the ki- netics depends both on agonist concentration and on solution ex- change rate. The nature of these dependences, and their interaction will be discussed, together with their possible mechanism. From the apparent solution exchange rate dependence it follows that measurements of the intrinsic kinetic properties of the receptor may be problematic, and experimentally measured kinetics may misrepresent intrinsic kinetics of the receptor. Abbreviations: a7 nAChR, a7 subtype nicotinic acetylcholine receptor; PNU- 120596, 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea; SXT, 10e90% solution exchange time. * Corresponding author. Tel.: þ36 12109971; fax: þ36 12109423. E-mail address: mike@koki.hu (A. Mike). Contents lists available at ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm 0028-3908/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2014.01.034 Neuropharmacology 81 (2014) 101e115