Jeremias Corradi

Single-channel and structural foundations of neuronal α7 acetylcholine receptor potentiation.

Potentiation of neuronal nicotinic acetylcholine receptors by exogenous ligands is a promising strategy for treatment of neurological disorders including Alzheimers disease and schizophrenia. To gain insight into molecular mechanisms underlying potentiation, we examined ACh-induced single-channel currents through the human neuronal α7 acetylcholine receptor in the presence of the α7-specific potentiator PNU-120596 (PNU). Compared to the unusually brief single-channel opening episodes elicited by agonist alone, channel opening episodes in the presence of agonist and PNU are dramatically prolonged. Dwell time analysis reveals that PNU introduces two novel components into open time histograms, indicating at least two degrees of PNU-induced potentiation. Openings of the longest potentiated class coalesce into clusters whose frequency and duration change over a narrow range of PNU concentration. At PNU concentrations approaching saturation, these clusters last up to several minutes, prolonging the submillisecond α7 opening episodes by several orders of magnitude. Mutations known to reduce PNU potentiation at the whole-cell level still give rise to multisecond-long single-channel clusters. However mutation of five residues lining a cavity within each subunits transmembrane domain abolishes PNU potentiation, defining minimal structural determinants of PNU potentiation.

Structural Basis of Activation of Cys-Loop Receptors: the Extracellular–Transmembrane Interface as a Coupling Region

Cys-loop receptors mediate rapid transmission throughout the nervous system by converting a chemical signal into an electric one. They are pentameric proteins with an extracellular domain that carries the transmitter binding sites and a transmembrane region that forms the ion pore. Their essential function is to couple the binding of the agonist at the extracellular domain to the opening of the ion pore. How the structural changes elicited by agonist binding are propagated through a distance of 50 Å to the gate is therefore central for the understanding of the receptor function. A step forward toward the identification of the structures involved in gating has been given by the recently elucidated high-resolution structures of Cys-loop receptors and related proteins. The extracellular–transmembrane interface has attracted attention because it is a structural transition zone where β-sheets from the extracellular domain merge with α-helices from the transmembrane domain. Within this zone, several regions form a network that relays structural changes from the binding site toward the pore, and therefore, this interface controls the beginning and duration of a synaptic response. In this review, the most recent findings on residues and pairwise interactions underlying channel gating are discussed, the main focus being on the extracellular–transmembrane interface.

Functional Relationships between Agonist Binding Sites and Coupling Regions of Homomeric Cys-Loop Receptors

Each subunit in a homopentameric Cys-loop receptor contains a specialized coupling region positioned between the agonist binding domain and the ion conductive channel. To determine the contribution of each coupling region to the stability of the open channel, we constructed a receptor subunit (α7-5-HT3A) with both a disabled coupling region and a reporter mutation that alters unitary conductance, and coexpressed normal and mutant subunits. The resulting receptors show single-channel current amplitudes that are quantized according to the number of reporter mutations per receptor, allowing correlation of the number of intact coupling regions with mean open time. We find that each coupling region contributes an equal increment to the stability of the open channel. However, by altering the numbers and locations of active coupling regions and binding sites, we find that a coupling region in a subunit flanked by inactive binding sites can still stabilize the open channel. We also determine minimal requirements for channel opening regardless of stability and find that channel opening can occur in a receptor with one active coupling region flanked by functional binding sites or with one active binding site flanked by functional coupling regions. The overall findings show that, whereas the agonist binding sites contribute interdependently and asymmetrically to open-channel stability, the coupling regions contribute independently and symmetrically.

Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating

The nicotinic receptor (AChR) is a pentamer of homologous subunits with an α2βεδ composition in adult muscle. Each subunit contains four transmembrane domains (M1–M4). Position 15′ of the M1 domain is phenylalanine in α subunits while it is isoleucine in non-α subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant α subunit (αF15′I) as well as those containing the reverse mutations in the non-α subunits (βI15′F, δI15′F, and εI15′F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the β subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of βI15′F AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant of the diliganded receptor, and a significant increased opening in the absence of agonist. Mutations at βI15′ showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an ∼70% closed-state-like environment for the β15′ position at the transition state of gating. The overall results identify position 15′ as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.

Differential contribution of subunit interfaces to α9α10 nicotinic acetylcholine receptor function

Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.

5-HT3 Receptor Brain-Type B-Subunits are Differentially Expressed in Heterologous Systems

Genes for five different 5-HT3 receptor subunits have been identified. Most of the subunits have multiple isoforms, but two isoforms of the B subunits, brain-type 1 (Br1) and brain-type 2 (Br2) are of particular interest as they appear to be abundantly expressed in human brain, where 5-HT3B subunit RNA consists of approximately 75% 5-HT3Br2, 24% 5-HT3Br1, and <1% 5-HT3B. Here we use two-electrode voltage-clamp, radioligand binding, fluorescence, whole cell, and single channel patch-clamp studies to characterize the roles of 5-HT3Br1 and 5-HT3Br2 subunits on function and pharmacology in heterologously expressed 5-HT3 receptors. The data show that the 5-HT3Br1 transcriptional variant, when coexpressed with 5-HT3A subunits, alters the EC50, nH, and single channel conductance of the 5-HT3 receptor, but has no effect on the potency of competitive antagonists; thus, 5-HT3ABr1 receptors have the same characteristics as 5-HT3AB receptors. There were some differences in the shapes of 5-HT3AB and 5-HT3ABr1 receptor responses, which were likely due to a greater proportion of homomeric 5-HT3A versus heteromeric 5-HT3ABr1 receptors in the latter, as expression of the 5-HT3Br1 compared to the 5-HT3B subunit is less efficient. Conversely, the 5-HT3Br2 subunit does not appear to form functional channels with the 5-HT3A subunit in either oocytes or HEK293 cells, and the role of this subunit is yet to be determined.

Stoichiometry for Activation of Neuronal α7 Nicotinic Receptors

Neuronal α7 nicotinic receptors elicit rapid calcium influx in response to acetylcholine (ACh) or its product choline. They contribute to cognition, synaptic plasticity, and neuroprotection, and have been implicated in neurodegenerative and neuropsychiatric disorders. α7, however, often localizes distal to sites of nerve-released ACh, binds ACh with low affinity, and thus elicits its biological response with low agonist occupancy. To assess function of α7 when neurotransmitter occupies fewer than five of its identical binding sites, we measured open-channel lifetime of individual receptors in which four of the five ACh binding-sites were disabled.

Number of Extracellular-Transmembrane Interfaces Required for Activation of Homomeric Cys-Loop Receptors

Each subunit in a homo-pentameric Cys-loop receptor contains a specialized transduction zone located at the extracellular-transmembrane interface that links the ligand binding domain to the ion conductive channel. To determine the contribution of each transduction zone to stability of the open channel, we constructed a subunit with both a disabled transduction zone and a reporter mutation that alters unitary conductance, and co-expressed mutant and normal subunits. The resulting receptors show single channel current amplitudes that are quantized according to the number of reporter mutations per receptor, allowing correlation of mean open time with the number of intact transduction zones. We find that each transduction zone contributes an equal increment to the stability of the open channel. However by combining subunits with either disabled agonist binding sites or transduction zones, we find that although each binding site is formed by a pair of subunits, detectable channel opening requires an intact transduction zone in both subunits. By manipulating the numbers and locations of transduction zones and binding sites, we find that a transduction zone in a subunit at an inactive binding site can still stabilize the open channel. The findings show that although the agonist binding sites and transduction zones contribute allosterically to open channel stability, their stoichiometry and positioning requirements are distinct.

A Novel Mechanism for the Inhibitory Action of Hydrocortisone at 5-HT3A Receptor

The 5-HT3A receptor is a member of the Cys-loop family of ligand-gated ion channels. Due to its low conductance, analysis of this receptor has been restricted to the macroscopic level. We introduced mutations in the 5-HT3A subunit to obtain a high-conductance form so that single channels can be detected. We studied the actions of the neuroactive steroid, hydrocortisone (HC), in the high-conductance form of the 5-HT3A receptor. Channel activity elicited by 1 μM 5-HT appears as opening events of 4.6±0.4 pA (−70 mV) forming bursts, which coalesce into long clusters. A minor population of lower amplitude events (∼2.8 pA) is observed, which corresponds to 0-10 % of the total events in all recordings. HC produces a concentration-dependent reduction in the duration of bursts and of the slowest open component (from ∼100 ms in the control to ∼3.6 ms at 400 μM HC), which can be explained on the basis of a slow block mechanism. Interestingly, amplitude histograms reveal a concentration-dependent increase in the relative area of the low-amplitude component without changes in its mean value. At 400 μM HC, the low-amplitude events correspond to 40 % of the total events. This channel population shows an amplitude of 2.8±0.5 pA (−70 mV). Macroscopic currents elicited by 5-HT in the presence of HC show reduced peak currents (∼50% at 400 μM HC) and increased decay rates compared to those recorded in the absence of the steroid. Taken together, our results reveal that hydrocortisone negatively modulates 5-HT3A receptors and show a novel mechanism which involves the stabilization of a subconductance state.