Orestes Quesada

Biophysical and Ion Channel Functional Characterization of the Torpedo californica Nicotinic Acetylcholine Receptor in Varying Detergent-Lipid Environments

The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, high-resolution structural studies have been hampered by the lack of mechanistic molecular models that describe how detergents influence membrane protein stability and function. Furthermore, elucidation of the dynamic detergent–lipid–protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examines the effects of nine detergents on: (1) nAChR-lipid composition (gas chromatography with flame ionization; GC-FID and/or mass selective detectors; GC-MSD), (2) stability and aggregation state (analytical size exclusion chromatography; A-SEC and electron microscopy; EM) and (3) ion channel function (planar lipid bilayers). Detergent solubilization of nAChR-enriched membranes did not result in significant native lipid depletion or destabilization. Upon purification, native lipid depletion occurred in all detergents, with lipid-analogue detergents CHAPS {(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate}, FC-12 (n-dodecylphosphocholine) and sodium cholate (3α,7α,12α-trihydroxy-5β-cholan-24-oic acid) maintaining stability and supporting ion channel function, and non-lipid-analogue detergents Cymal-6 (6-cyclohexyl-1-hexyl-β-D-maltoside), DDM (n-dodecyl-β-D-maltopyranoside), LDAO (lauryldimethylamine-N-oxide) and OG (n-octyl-β-d-glucopyranoside) decreasing stability and significantly reducing or completely suppressing ion channel function. Anapoe-C12E9 (polyoxyethylene-[9]-dodecyl ether) and BigCHAP (N,N’-bis-[3-d-gluconamidopropyl] cholamide) retained residual amounts of native lipid, maintaining moderate stability and ion channel function compared to lipid-analogue detergents. Therefore, the nAChR can be stable and functional in lipid-analogue detergents or in detergents that retain moderate amounts of residual native lipids, but not in non-lipid-analogue detergents.

The α7-nicotinic receptor is upregulated in immune cells from HIV-seropositive women: consequences to the cholinergic anti-inflammatory response.

Antiretroviral therapy partially restores the immune system and markedly increases life expectancy of HIV‐infected patients. However, antiretroviral therapy does not restore full health. These patients suffer from poorly understood chronic inflammation that causes a number of AIDS and non‐AIDS complications. Here we show that chronic inflammation in HIV+ patients may be due to the disruption of the cholinergic anti‐inflammatory pathway by HIV envelope protein gp120IIIB. Our results demonstrate that HIV gp120IIIB induces α7 nicotinic acetylcholine receptor (α7) upregulation and a paradoxical proinflammatory phenotype in macrophages, as activation of the upregulated α7 is no longer capable of inhibiting the release of proinflammatory cytokines. Our results demonstrate that disruption of the cholinergic‐mediated anti‐inflammatory response can result from an HIV protein. Collectively, these findings suggest that HIV tampering with a natural strategy to control inflammation could contribute to a crucial, unresolved problem of HIV infection: chronic inflammation.

Effects of lipid-analog detergent solubilization on the functionality and lipidic cubic phase mobility of the Torpedo californica nicotinic acetylcholine receptor.

Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized β2-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility.

Expression of CHRFAM7A and CHRNA7 in neuronal cells and postmortem brain of HIV-infected patients: considerations for HIV-associated neurocognitive disorder

Despite the recent advances in antiretroviral therapy, human immunodeficiency virus type 1 (HIV-1) remains a global health threat. HIV-1 affects the central nervous system by releasing viral proteins that trigger neuronal death and neuroinflammation, and promotes alterations known as HIV-associated neurocognitive disorders (HAND). This disorder is not fully understood, and no specific treatments are available. Recently, we demonstrated that the HIV-1 envelope protein gp120IIIB induces a functional upregulation of the α7-nicotinic acetylcholine receptor (α7) in neuronal cells. Furthermore, this upregulation promotes cell death that can be abrogated with receptor antagonists, suggesting that α7 may play an important role in the development of HAND. The partial duplication of the gene coding for the α7, known as CHRFAM7A, negatively regulates α7 expression but its role in HIV infection has not been studied. Hence, we studied both CHRNA7 and CHRFAM7A regulation patterns in various gp120IIIB in vitro conditions. In addition, we measured CHRNA7 and CHRFAM7A expression levels in postmortem brain samples from patients suffering from different stages of HAND. Our results demonstrate the induction of CHRNA7 expression accompanied by a significant downregulation of CHRFAM7A in neuronal cells when exposed to pathophysiological concentrations of gp120IIIB. Our results suggest a dysregulation of CHRFAM7A and CHRNA7 expressions in the basal ganglia from postmortem brain samples of HIV+ subjects and expand the current knowledge about the consequences of HIV infection in the brain.

The alpha7-nicotinic receptor contributes to gp120-induced neurotoxicity: implications in HIV-associated neurocognitive disorders

Currently, there are no specific therapies to treat HIV-1 associated neurocognitive disorders (HAND). The HIV-1 envelope, gp120, induces neuropathological changes similar to those in HAND patients; furthermore, it triggers an upregulation of the α7-nicotinic acetylcholine receptor (α7-nAChR), facilitating intracellular calcium overload and neuronal cell death. Using a gp120IIIB-transgenic mouse (gp120-tgm) model, we demonstrate that α7-nAChRs are upregulated on striatal neurons. Activation of α7-nAChRs leads to an increase in both intracellular calcium and percentage of apoptotic cells, which can be abrogated by antagonizing the receptor, suggesting a role for α7-nAChRs in gp120-induced neurotoxicity. Moreover, we demonstrate for the first time that gp120-tgm have learning deficiencies on a striatum-dependent behavioral task. They also show locomotor deficiencies, which improved with α7-nAChR antagonists, further supporting a role for this receptor in gp120-induced neurotoxicity. Together, these results uncover a new mechanism through which gp120-induced modulation of α7-nAChRs in the striatum can contribute to HAND development.

A Panel of Slow-Channel Syndrome Mice Reveals a Unique Locomotor Behavioral Signature

Muscle nicotinic acetylcholine receptor (nAChR) mutations can lead to altered channel kinetics and neuromuscular junction degeneration, a neurodegenerative disorder collectively known as slow-channel syndrome (SCS). A multivariate analysis using running wheels was used to generate activity profiles for a variety of SCS models, uncovering unique locomotor patterns for the different nAChR mutants. Particularly, the αL251T and ɛL269F mutations exhibit decreased event distance, duration, and velocity over a period of 24 hours. Our approach suggests a robust relationship between the pathophysiology of SCS and locomotor activity.

Lateral diffusion, function, and expression of the slow channel congenital myasthenia syndrome αC418W nicotinic receptor mutation with changes in lipid raft components.

Background: αC418W nAChR is the first lipid-exposed mutation identified in a patient that causes slow channel congenital myasthenia syndrome (SCCMS). Results: The αC418W nAChR is highly immobile and sensitive to lipid raft components. Conclusion: Cholesterol and CAV-1 modulate the function and dynamics of αC418W nAChRs. Significance: Understanding the interplay between cholesterol, CAV-1, and nAChRs is crucial for developing potential therapeutic treatments for this disease. Lipid rafts, specialized membrane microdomains in the plasma membrane rich in cholesterol and sphingolipids, are hot spots for a number of important cellular processes. The novel nicotinic acetylcholine receptor (nAChR) mutation αC418W, the first lipid-exposed mutation identified in a patient that causes slow channel congenital myasthenia syndrome was shown to be cholesterol-sensitive and to accumulate in microdomains rich in the membrane raft marker protein caveolin-1. The objective of this study is to gain insight into the mechanism by which lateral segregation into specialized raft membrane microdomains regulates the activable pool of nAChRs. We performed fluorescent recovery after photobleaching (FRAP), quantitative RT-PCR, and whole cell patch clamp recordings of GFP-encoding Mus musculus nAChRs transfected into HEK 293 cells to assess the role of cholesterol and caveolin-1 (CAV-1) in the diffusion, expression, and functionality of the nAChR (WT and αC418W). Our findings support the hypothesis that a cholesterol-sensitive nAChR might reside in specialized membrane microdomains that upon cholesterol depletion become disrupted and release the cholesterol-sensitive nAChRs to the pool of activable receptors. In addition, our results in HEK 293 cells show an interdependence between CAV-1 and αC418W that could confer end plates rich in αC418W nAChRs to a susceptibility to changes in cholesterol levels that could cause adverse drug reactions to cholesterol-lowering drugs such as statins. The current work suggests that the interplay between cholesterol and CAV-1 provides the molecular basis for modulating the function and dynamics of the cholesterol-sensitive αC418W nAChR.

Uncovering the lipidic basis for the preparation of functional nicotinic acetylcholine receptor detergent complexes for structural studies.

This study compares the lipid composition, including individual phospholipid molecular species of solubilized nAChR detergent complexes (nAChR-DCs) with those of the bulk lipids from their source, Torpedo californica (Tc) electric tissue. This lipidomic analysis revealed seventy-seven (77) phospholipid species in the Tc tissue. Analysis of affinity-purified nAChR-DCs prepared with C-12 to C-16 phospholipid analog detergents alkylphosphocholine (FC) and lysofoscholine (LFC) demonstrated that nAChR-DCs prepared with FC12, LFC14, and LFC16 contained >60 phospholipids/nAChR, which was more than twice of those prepared with FC14, FC16, and LFC12. Significantly, all the nAChR-DCs lacked ethanolamine and anionic phospholipids, contained only four cholesterol molecules, and a limited number of phospholipid molecular species per nAChR. Upon incorporation into oocytes, FC12 produce significant functionality, whereas LFC14 and LFC16 nAChR-DCs displayed an increased functionality as compared to the crude Tc membrane. All three nAChR-DCs displayed different degrees of alterations in macroscopic activation and desensitization kinetics.

Heterogeneous Inhibition in Macroscopic Current Responses of Four Nicotinic Acetylcholine Receptor Subtypes by Cholesterol Enrichment

The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. In addition, the nAChR is also found in the electric organs of electric rays (e.g., the genus Torpedo). Cholesterol, which is a key lipid for maintaining the correct functionality of membrane proteins, has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs expressed in a model membrane with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P ratio in Xenopus laevis oocytes expressing the neuronal α7, α4β2, muscle-type, and Torpedo californica nAChRs in their macroscopic current responses. Using the two-electrode voltage clamp technique, it was found that the neuronal α7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle-type nAChR. The peak current versus C/P profiles during enrichment display different behaviors; α7 and Torpedo nAChRs display a hyperbolic decay with two clear components, whereas muscle-type and α4β2 nAChRs display simple monophasic decays with different slopes. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration produces a remarkable reduction in the macroscopic current responses of the neuronal α7 and Torpedo nAChRs functionality, whereas the muscle nAChR appears to be the most resistant to cholesterol inhibition among all four nAChR subtypes. Overall, the present study demonstrates differential profiles for cholesterol inhibition among the different types of nAChR to physiological cholesterol increments in the plasmatic membrane. This is the first study to report a cross-correlation analysis of cholesterol sensitivity among different nAChR subtypes in a model membrane.