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Both bioactive sphingolipids and Sigma-1 receptor (S1R) chaperones occur ubiquitously in mammalian cell membranes. Endogenous compounds that regulate the S1R are important for controlling S1R responses to cellular stress. Herein, we interrogated the S1R in intact Retinal Pigment Epithelial cells (ARPE-19) with the bioactive sphingoid base, sphingosine (SPH), or the pain-provoking dimethylated SPH derivative, N,N'-dimethylsphingosine (DMS). As informed by a modified native gel approach, the basal and antagonist (BD-1047)-stabilized S1R oligomers dissociated to protomeric forms in the presence of SPH or DMS (PRE-084 as control). We, thus, posited that SPH and DMS are endogenous S1R agonists. Consistently, in silico docking of SPH and DMS to the S1R protomer showed strong associations with Asp126 and Glu172 in the cupin beta barrel and extensive van der Waals interactions of the C18 alkyl chains with the binding site including residues in helices 4 and 5. Mean docking free energies were 8.73–8.93 kcal/mol for SPH and 8.56–8.15 kcal/mol for DMS, and calculated binding constants were ~40 nM for SPH and ~120 nM for DMS. We hypothesize that SPH, DMS, and similar sphingoid bases access the S1R beta barrel via a membrane bilayer pathway. We further propose that the enzymatic control of ceramide concentrations in intracellular membranes as the primary sources of SPH dictates availability of endogenous SPH and DMS to the S1R and the subsequent control of S1R activity within the same cell and/or in cellular environments. [ABSTRACT FROM AUTHOR]

The [sigma]-receptor, a broadly distributed integral membrane protein with a novel structure, is known to modulate various voltage-gated [K.sup.+] and [Ca.sup.2+] channels through a mechanism that involves neither G proteins nor phosphorylation. The present study investigated the modulation of the heart voltage-gated [Na.sup.+] channel ([Na.sub.v]1.5]) by [sigma]-receptors. The [[sigma].sub.1]-receptor ligands [SKF-10047 and (+)-pentazocine] and [[sigma].sub.1]/[[sigma].sub.2]-receptor ligands (haloperidol and ditolylguanidine) all reversibly inhibited [Na.sub.v]l.5 channels to varying degrees in human embryonic kidney 293 (HEK-293) cells and COS-7 cells, but the [[sigma].sub.1]-receptor ligands were less effective in COS-7 cells. The same four ligands also inhibited [Na.sup.+] current in neonatal mouse cardiac myocytes. In [[sigma].sub.1]-receptor knockout myocytes, the [[sigma].sub.1]-receptor-specific ligands were far less effective in modulating [Na.sup.+] current, but the [[sigma].sub.1]/[[sigma].sub.1]-receptor ligands modulated [Na.sup.+] channels as well as in wild type. Photolabeling with the [[sigma].sub.1]-receptor photoprobe [[sup.125]I]-iodoazidococaine demonstrated that [[sigma].sub.1] receptors were abundant in heart and HEK-293 cells, but scarce in COS-7 cells. This difference was consistent with the greater efficacy of [[sigma].sub.1]-receptor-specific ligands in HEK-293 cells than in COS-7 cells. [sigma]-Receptors modulated [Na.sup.+] channels despite the omission of GTP and ATP from the patch pipette solution, [sigma]-Receptor-mediated inhibition of [Na.sup.+] current had little if any voltage dependence and produced no change in channel kinetics. [Na.sup.+] channels represent a new addition to the large number of voltage-gated ion channels modulated by [sigma]-receptors. The modulation of [Na.sub.v]1.5 channels by [sigma]-receptors in the heart suggests an important pathway by which drugs can alter cardiac excitability and rhythmicity. heart muscle; haloperidol

The retinal phosphodiesterase (PDE6) inhibitory [gamma]-subunit (PD[E.sub.[gamma]],) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic [alpha][beta]-subunits of PDE6 and the [alpha]-subunit of transducin ([[alpha].sub.t]). Detailed structural analysis of PD[E.sub.[gamma]] has been hampered by its intrinsic disorder. We present here the NMR solution structure of PD[E.sub.[gamma]], which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PD[E.sub.[gamma]46-87] when bound to at in the transitionstate complex. NMR mapping of the interaction between PD[E.sub.[gamma]46-87] and the chimeric PDE5/6 catalytic domain confirmed that Cterminal residues 74-87 of PD[E.sub.[gamma]] are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to at, is left free at this stage. These results indicate that the interaction between PD[E.sub.[gamma]] and [[alpha].sub.t] during the phototransduction cascade involves the selection of preconfigured transient conformations. NMR spectroscopy | protein recognition | transient structure | visual transduction

Research has been conducted on the vesicle monoamine transporter which gathers monoamine neurotransmitter into synaptic vesicles. The role of the naturally occurring disulfide bond in the monoamine transport into vesicles has been investigated and the details are reported.

The synthesis and characterization of various benzophenone- and fluorenone-based photoprobes for the beta(sub.2)-adrenergic receptor (beta(sub.2)AR) are described. Alprenolol protectable photoaffinity labeling of the beta(sub.2)AR in membranes has been used to show that ((super.125)I)IamF, one of the labels, can be used to identify the amino acid(s) in the beta(sub.2)AR that interact with antagonist pharmacophores.