Exploring the positive allosteric modulation of human α7 nicotinic receptors from a single-channel perspective

Highlights

• Potentiation of α7 is analyzed at the single-channel level.

• Type I and II PAMs prolong open-channel lifetime and activation episodes.

• Temperature sensitivity and structural determinants differ between PAM types.

• Some though not all type I PAMs share structural determinants of type II PAMs.

Abstract

Enhancement of α7 nicotinic receptor (nAChR) function by positive allosteric modulators (PAMs) is a promising therapeutic strategy to improve cognitive deficits. PAMs have been classified only on the basis of their macroscopic effects as type I, which only enhance agonist-induced currents, and type II, which also decrease desensitization and reactivate desensitized nAChRs. To decipher the molecular basis underlying these distinct activities, we explored the effects on single-α7 channel currents of representative members of each type and of less characterized compounds. Our results reveal that all PAMs enhance open-channel lifetime and produce episodes of successive openings, thus indicating that both types affect α7 kinetics. Different PAM types show different sensitivity to temperature, suggesting different mechanisms of potentiation. By using a mutant α7 receptor that is insensitive to the prototype type II PAM (PNU-120596), we show that some though not all type I PAMs share the structural determinants of potentiation. Overall, our study provides novel information on α7 potentiation, which is key to the ongoing development of therapeutic compounds.

Introduction

α7 nicotinic receptors (nAChRs) are widely distributed in the brain, especially in the hippocampus, thalamus, and cortex (Albuquerque et al., 2009). They contribute to cognition, sensory information processing, attention, working memory, and reward pathways. Decline or alterations of cholinergic signaling involving α7 have been implicated in various neurological diseases, such as schizophrenia, epilepsy, and Alzheimer's disease (Dani and Bertrand, 2007, Dineley et al., 2015, Hurst et al., 2013, Thomsen et al., 2010, Wallace and Bertrand, 2013, Wallace and Porter, 2011). Selective α7 agonists are currently being developed for the treatment of memory impairment in patients with schizophrenia and Alzheimer's disease (Fan et al., 2015, Freedman, 2014, Wallace and Porter, 2011). An alternative approach to increase α7 function is the use of selective positive allosteric modulators (PAMs) (Arias, 2010, Uteshev, 2014, Williams et al., 2011a). Allosteric ligands have several pharmacological advantages over orthosteric ligands including maintenance of the normal spatial and temporal pattern of endogenous neurotransmission and higher receptor subtype selectivity, resulting, at least hypothetically, in high clinical efficacy with minimal adverse effects (Uteshev, 2014).

PAMs have been classified on the basis of their macroscopic effects as type I (e.g., 5-HI, NS-1738) or type II (e.g., PNU-120596). Based on their macroscopic effects it has been postulated that type I PAMs only enhance agonist-induced currents without affecting macroscopic current kinetics, whereas type II PAMs also delay desensitization and reactivate desensitized receptors (Arias, 2010, Bertrand and Gopalakrishnan, 2007, Williams et al., 2011a). The microscopic origin of these profiles remains unclear for most PAMs and its elucidation requires high-resolution single-channel recordings.

We have recently synthesized a series of compounds, named as PAM-2, PAM-3, and PAM-4 and shown that they act as selective α7 PAMs (Arias et al., 2011). Initial Ca²⁺ influx experiments show that PAM-2 reactivates desensitized α7, suggesting that it is a putative type II PAM (Targowska-Duda et al., 2014). The potential clinical importance of these PAMs is based on experimental results in rodents revealing that PAM-2 produces antidepressant-like (Targowska-Duda et al., 2014, Arias et al., 2015), pro-cognitive (Potasiewicz et al., 2015), and nociceptive and anti-inflammatory activities (Bagdas et al., 2015).

Considering the wide spectrum of potential clinical uses of PAMs, understanding the underlying molecular mechanism of potentiation at human α7 is urgent. Still, studying α7 at the molecular level is complex due to its low open probability and fast kinetics, high-resolution single-channel recordings being therefore required to collect accurate information (Bouzat et al., 2008). We therefore performed a thorough evaluation at the single-channel current level of the activity of prototypic type I and type II PAMs and of the less characterized compounds (PAM-2, -3 and -4). Overall, by examining PAM activities from a different perspective, our results provide novel information regarding the foundation of α7 potentiation, which is required for understanding the potential consequences at the cell and clinical levels.