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 (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid) maintaining stability and supporting ion channel function, and non-lipid-analogue detergents Cymal-6 (6-cyclohexyl-1-hexyl-beta-D-maltoside), DDM (n-dodecyl-beta-D-maltopyranoside), LDAO (lauryldimethylamine-N-oxide) and OG (n-octyl-beta-d-glucopyranoside) decreasing stability and significantly reducing or completely suppressing ion channel function. Anapoe-C(12)E(9 )(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.

Testing forelimb placing "across the midline" reveals distinct, lesion-dependent patterns of recovery in rats.

We describe a new test of vibrissae-elicited forelimb placing ability that allows testing of sensorimotor integration across the midline. Rats were given unilateral brain lesions using one of three methods: (1) middle cerebral artery occlusion (MCAo) causing significant damage to the cortex and striatum, (2) aspiration lesions to remove tissue from the sensorimotor cortex, and (3) infusions of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, producing a parkinsonian syndrome. Application of the new test to these animals revealed that with some lesion types, the ability of vibrissae on the unimpaired side of the body to trigger placing in the functionally impaired forelimb recovers before vibrissae on the impaired side can elicit placing. This occurs despite the lack of any apparent vibrissae sensory deficit, since the contralesional vibrissae maintained the ability to trigger placing in the unimpaired forelimb in all lesions studied. Chronically, MCAo-lesioned rats do not place the impaired forelimb upon stimulation of the impaired-side vibrissae, but do place if the vibrissae on the good side are stimulated (i.e., when the placing is triggered "across the midline"). This is in contrast to 6-OHDA-lesioned rats which, consistent with parkinsonian akinesia, cannot place the impaired limb regardless of sensory trigger. Also, differences in the pattern of recovery between MCAo- and aspiration-lesioned rats suggest a possible anatomical substrate for cross-midline placing ability and its recovery. Unlike other tests, cross-midline placing methods can readily distinguish between severe stroke and severe parkinsonism in rats.