Inhibition of calpain and caspase-3 prevented apoptosis and preserved electrophysiological properties of voltage-gated and ligand-gated ion channels in rat primary cortical neurons exposed to glutamate.

Glutamate toxicity in traumatic brain injury, ischemia, and Huntington's disease causes cortical neuron death and dysfunction. We tested the efficacy of calpain and caspase-3 inhibitors alone and in combination to prevent neuronal death and preserve electrophysiological functions in rat primary cortical neurons following glutamate exposure. Cortical neurons exposed to 0.5 microM glutamate for 24 h committed mostly apoptotic death as determined by Wright staining and ApopTag assay. Levels of expression, formation of active forms, and activities of calpain and caspase-3 were increased following glutamate exposure. Also, in situ double labeling identified conformationally active caspase-3-p20 fragment and chromatin condensation in apoptotic neurons. Pretreatment of cortical neurons with 0.2 microM N-benzyloxylcarbonyl-Leu-Nle-aldehyde (calpain-specific inhibitor) and 100 microM N-benzyloxylcarbonyl-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethyl ketone (caspase-3-specific inhibitor) provided strong neuroprotection. Standard patch-clamp techniques were used to measure the whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors. The lack of a change in capacitance indicated that neurons treated with inhibitor(s) plus glutamate did not undergo apoptotic shrinkage and maintained the same size as the control neurons. Whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors were similar in amplitude and activation/inactivation kinetics for cells untreated and treated with inhibitor(s) and glutamate. Spontaneous synaptic activity as observed by miniature end-plate currents was also similar. Prevention of glutamate-induced apoptosis by calpain and caspase-3 inhibitors preserved normal activities of crucial ion channels such as Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors in neurons. Our studies strongly imply that calpain and caspase-3 inhibitors may also provide functional neuroprotection in the animal models of traumatic brain injury and neurodegenerative diseases.

Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety.

Knock-in mice were generated that harbored a leucine-to-serine mutation in the alpha4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.

Oxidative stress and Ca2+ influx upregulate calpain and induce apoptosis in PC12 cells.

Calpain, a Ca2+-dependent cysteine protease, has previously been implicated in apoptosis or programmed cell death (PCD) in immune cells. Although oxidative stress and intracellular free Ca2+ are involved in neurodegenerative diseases, the mechanism of neuronal cell death in the central nervous system (CNS) due to these agents has not yet been defined. To explore a possible role for calpain in neuronal PCD under oxidative stress and Ca2+ influx, we examined the effects of H2O2 and A23187 on PC12 cells. Treatments caused PCD (light microscopy and TUNEL assay) with altered mRNA expression (RT-PCR) of bax (pro-apoptotic) and bcl-2 (anti-apoptotic) genes, resulting in a high bax/bcl-2 ratio. Control cells expressed 1.3-fold more microcalpain (requiring microM Ca2+) than mcalpain (requiring mM Ca2+). Expression of mcalpain was significantly increased following exposure to oxidative stress and Ca2+ influx. The mRNA levels of calpastatin (endogenous calpain inhibitor) and beta-actin (house-keeping) genes were not changed. Western analysis indicated degradation of 68 kDa neurofilament protein (NFP), a calpain substrate. Pretreatment of cells with MDL28170 (a cell permeable and selective inhibitor of calpain) prevented increase in bax/bcl-2 ratio, upregulation of calpain, degradation of 68 kDa NFP, and occurrence of PCD. These results suggest a role for calpain in PCD of PC12 cells due to oxidative stress and Ca2+ influx.

In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system.

A general method for the incorporation of unnatural amino acids into ion channels and membrane receptors using a Xenopus oocyte expression system has been described. A large number of unnatural amino acids have been incorporated into the nAChR, GIRK, and Shaker K+ channels. Continuing efforts focus on incorporating unnatural amino acids that differ substantially from the natural amino acids, for example, residues that include fluorophores. In addition, we are addressing the feasibility of incorporating unnatural amino acids into ion channels and membrane receptors in mammalian cells.

Dose-response relations for unnatural amino acids at the agonist binding site of the nicotinic acetylcholine receptor: tests with novel side chains and with several agonists.

Structure-function relations in the nicotinic acetylcholine receptor are probed using a recently developed method based on chemical synthesis of nonsense suppressor tRNAs with unnatural amino acid residues, site-directed incorporation at nonsense codons in Xenopus laevis oocytes, and electrophysiological measurements. A broad range of unnatural amino acids, as many as 14 at a given site, are incorporated at three sites, alpha 93, alpha 190, and alpha 198, all of which are tyrosine in the wild-type receptor and are thought to contribute to the agonist binding site. Confirming and expanding upon earlier studies using conventional mutagenesis, the three tyrosines are shown to be in substantially different structural microenvironments. In particular, a crucial role is established for the hydroxyl group of alpha Tyr93, whereas a variety of substituents are functional at the analogous position of alpha Tyr198. Interestingly, consideration of three different agonists (acetylcholine, nicotine, and tetramethylammonium) does not discriminate between these two best-characterized binding site residues. In addition, double-mutation studies establish the independent effects of mutations at the pore region (second transmembrane region) and at the agonist binding site, and this observation leads to a novel strategy for adjusting EC50 values. These results establish the broad generality and great potential of the unnatural amino acid methodology for illuminating subtle structural distinctions in neuroreceptors and related integral membrane proteins.

An engineered Tetrahymena tRNAGln for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression.

A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology.

Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors.

In nicotinic acetylcholine receptors (nAChR), as well as glycine, GABAA (gamma-aminobutyric acid), serotonin (5-HT3), and GluCl glutamate receptors, a leucine residue at the approximate midpoint of the M2 transmembrane domain (the 9' position) is conserved across most known subunits. Structural data for the nAChR suggest that the Leu 9' residues occupy a 'kink' in each of the five M2 helices and point into the closed channel; in the opening step, the M2 helices rotate so that Leu 9' side chains no longer occlude the conduction pathway. Mutation of Leu 9' to one of several other residues slows desensitization and increases sensitivity to agonist. We have exploited the alpha 2 beta gamma delta stoichiometry of muscle nAChR to express receptors with ms* = 0 to 5 Leu 9'Ser mutated subunits. Strikingly, each Leu 9'Ser mutation shifts the dose-response relation for ACh to the left by approximately 10-fold; a nAChR with ms* = 4 is 10(4)-fold more sensitive than the wild type. The results suggest that each of the five Leu 9' residues participates independently and symmetrically in a key step in the structural transition between the closed and open states.

Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells.

The nonsense codon suppression method for unnatural amino acid incorporation has been applied to intact cells and combined with electrophysiological analysis to probe structure-function relations in the nicotinic acetylcholine receptor. Functional receptors were expressed in Xenopus oocytes when tyrosine and phenylalanine derivatives were incorporated at positions 93, 190, and 198 in the binding site of the alpha subunit. Subtle changes in the structure of an individual side chain produced readily detectable changes in the function of this large channel protein. At each position, distinct features of side chain structure dominated the dose-response relation, probably by governing the agonist-receptor binding.

Fluorescence studies on the interactions of myelin basic protein in electrolyte solutions.

This paper examines the influence of electrolytes on fluorescence spectral properties of the single tryptophanyl residue, Trp-115, within the 18.5-kDa species of myelin basic protein from bovine brain. Steady-state fluorescence spectra and intensities and time-correlated fluorescence lifetimes increased in the presence of increasing concentrations of mono- and divalent electrolytes (Li+, Na+, K+, Mg2+, Ca2+, Cl-, ClO4-, SO4(2-), and PO4(3-)). In all cases, the increases closely paralleled the ionic strength of the bulk aqueous medium and resembled that observed upon immersion of the protein in solutions of urea. This behavior was therefore concluded to reflect changes in the solution conformation of myelin basic protein. Bimolecular quenching of Trp-115 by acrylamide was rapid (10(9) M-1 s-1), approaching the diffusion limitation, and markedly dependent on the viscosity of the bulk aqueous medium. Rotational depolarization of myelin basic protein was rapid (phi less than or equal to 1 ns), occurring at rates exceeding those predicted for a rigid particle of revolution, and markedly dependent on the viscosity of the surrounding medium. Whereas the bimolecular quenching constants were unaltered in the presence of electrolytes, rotational depolarization of myelin basic protein underwent substantial slowing as indicated by the appearance of an additional decay component characterized by a correlation time of 5-10 ns. These studies indicate that Trp-115 of myelin basic protein is readily accessible to the bulk aqueous medium and is associated with a highly mobile segment of the protein. The slowing of rotational depolarization upon immersion of myelin basic protein in electrolyte solutions is consistent with an electrolyte-induced self-association of myelin basic protein molecules and indicates a relationship between the lability of solution conformation on the one hand and the capacity for self-association on the other.

Estrogen treatment increases the density of D1 dopamine receptors in the rat striatum.

Adult, male rats were treated with 17 beta-estradiol valerate by s.c. injection of 125 micrograms/rat. Six days later the density and affinity of striatal D1 dopamine (DA) receptors were determined. Estrogen treatment significantly increased the density of D1 DA receptors without altering their affinity. In vitro co-incubation with 17 beta-estradiol 17 beta-estradiol valerate at concentrations up to 1.0 microM did not alter binding to D1 DA receptors.

Site selectivity of fluorescent bisquaternary phenanthridinium ligands for acetylcholinesterase.

The synthesis of decidium and hexidium diiodides, their spectroscopic properties, and association with acetylcholinesterase from Torpedo californica are described and compared with those for propidium. Decidium, hexidium, and propidium, bisquaternary analogs of the fluorescent phenanthridinium ligand ethidium, contain 10, 6, and 3 methylene carbons, respectively, interposed between the exocyclic and endocyclic quaternary nitrogens. The three ligands exhibit linear competitive inhibition of enzyme carbamylation by N-methyl-7-dimethylcarbamoxyquinolinium. Dissociation constants for decidium, hexidium, and propidium are found by direct fluorescence titration to be 2.1 +/- 0.2 X 10(-8), 5.8 +/- 1.4 X 10(-7), and 3.7 +/- 0.4 X 10(-6) M, values in close accord with the inhibition constants obtained from kinetic analyses. Association of the three ligands is characterized by a stoichiometry of one fluorescent ligand per 80-kDa molecular weight subunit and occurs with respective 6.5-, 4.5-, and 3-fold increases in both quantum yield and fluorescence lifetime. Decidium and hexidium, in marked contrast with propidium, are dissociated by ligands selective for the active center and undergo pronounced reduction in affinity upon modification of the active center with pyrenebutyl methylphosphonofluoridate. Whereas the kinetics reveal no clear distinctions in inhibitory action of the three ligands, the fluorescence studies indicate that the alkyltrimethylammonium moiety of decidium and hexidium occludes the active center; propidium, in contrast, associates solely with the peripheral anionic site and does not occlude the active center. The temperature dependence of binding indicates that decidium association engenders a substantial increase (+55 eu) in entropy. The data indicate that the active center and peripheral anionic sites are separated by a crevice which can accommodate the hydrocarbon portion of extended n-alkyl cationic ligands, thereby affording entropic stabilization of complex formation. This stabilization is realized, however, only when the anionic subsite of the active center is not occluded, enabling electrostatic interaction between cationic ligand and the anionic active center.