Disruption of nNOS-PSD95 protein-protein interaction inhibits acute thermal hyperalgesia and chronic mechanical allodynia in rodents.

BACKGROUND AND PURPOSE: Post-synaptic density protein 95 (PSD95) contains three PSD95/Dosophilia disc large/ZO-1 homology domains and links neuronal nitric oxide synthase (nNOS) with the N-methyl-D-aspartic acid (NMDA) receptor. This report assesses the effects of disruption of the protein-protein interaction between nNOS and PSD95 on pain sensitivity in rodent models of hyperalgesia and neuropathic pain. EXPERIMENTAL APPROACH: We generated two molecules that interfered with the nNOS-PSD95 interaction: IC87201, a small molecule inhibitor; and tat-nNOS (residues 1-299), a cell permeable fusion protein containing the PSD95 binding domain of nNOS. We then characterized these inhibitors using in vitro and in vivo models of acute hyperalgesia and chronic allodynia, both of which are thought to require nNOS activation. KEY RESULTS: IC87201 and tat-nNOS (1-299) inhibited the in vitro binding of nNOS with PSD95, without inhibiting nNOS catalytic activity. Both inhibitors also blocked NMDA-induced 3',5'-cyclic guanosine monophosphate (cGMP) production in primary hippocampal cultures. Intrathecal administration of either inhibitor potently reversed NMDA-induced thermal hyperalgesia in mice. At anti-hyperalgesic doses, there was no effect on acute pain thresholds or motor coordination. Intrathecal administration of IC87201 and tat-nNOS also reversed mechanical allodynia induced by chronic constriction of the sciatic nerve. CONCLUSIONS AND IMPLICATIONS: nNOS-PSD95 interaction is important in maintaining hypersensitivity in acute and chronic pain. Disruption of the nNOS-PSD95 interaction provides a novel approach to obtain selective anti-hyperalgesic compounds.

The delta subunit of type 6 phosphodiesterase reduces light-induced cGMP hydrolysis in rod outer segments.

The delta subunit of the rod photoreceptor PDE has previously been shown to copurify with the soluble form of the enzyme and to solubilize the membrane-bound form (). To determine the physiological effect of the delta subunit on the light response of bovine rod outer segments, we measured the real time accumulation of the products of cGMP hydrolysis in a preparation of permeablized rod outer segments. The addition of delta subunit GST fusion protein (delta-GST) to this preparation caused a reduction in the maximal rate of cGMP hydrolysis in response to light. The maximal reduction of the light response was about 80%, and the half-maximal effect occurred at 385 nm delta subunit. Several experiments suggest that this effect was not due to the effects of delta-GST on transducin or rhodopsin kinase. Immunoblots demonstrated that exogenous delta-GST solubilized the majority of the PDE in ROS but did not affect the solubility of transducin. Therefore, changes in the solubility of transducin cannot account for the effects of delta-GST in the pH assay. The reduction in cGMP hydrolysis was independent of ATP, which indicates that it was not due to effects of delta-GST on rhodopsin kinase. In addition to the effect on cGMP hydrolysis, the delta-GST fusion protein slowed the turn-off of the system. This is probably due, at least in part, to an observed reduction in the GTPase rate of transducin in the presence of delta-GST. These results demonstrate that delta-GST can modify the activity of the phototransduction cascade in preparations of broken rod outer segments, probably due to a functional uncoupling of the transducin to PDE step of the signal transduction cascade and suggest that the delta subunit may play a similar role in the intact outer segment.

Binding of the delta subunit to rod phosphodiesterase catalytic subunits requires methylated, prenylated C-termini of the catalytic subunits.

PDE6 (type 6 phosphodiesterase) from rod outer segments consists of two types of catalytic subunits, alpha and beta; two inhibitory gamma subunits; and one or more delta subunits found only on the soluble form of the enzyme. About 70% of the phosphodiesterase activity found in rod outer segments is membrane-bound, and is thought to be anchored to the membrane through C-terminal prenyl groups. The recombinant delta subunit has been shown to solubilize the membrane-bound form of the enzyme. This paper describes the site and mechanism of this interaction in more detail. In isolated rod outer segments, the delta subunit was found exclusively in the soluble fraction, and about 30% of it did not coimmunoprecipitate with the catalytic subunits. The delta subunit that was bound to the catalytic subunits dissociated slowly, with a half-life of about 3.5 h. To determine whether the site of this strong binding was the C-termini of the phosphodiesterase catalytic subunits, peptides corresponding to the C-terminal ends of the alpha and beta subunits were synthesized. Micromolar concentrations of these peptides blocked the phosphodiesterase/delta subunit interaction. Interestingly, this blockade only occurred if the peptides were both prenylated and methylated. These results suggested that a major site of interaction of the delta subunit is the methylated, prenylated C-terminus of the phosphodiesterase catalytic subunits. To determine whether the catalytic subunits of the full-length enzyme are methylated in situ when bound to the delta subunit, we labeled rod outer segments with a tritiated methyl donor. Soluble phosphodiesterase from these rod outer segments was more highly methylated (4.5 +/- 0.3-fold) than the membrane-bound phosphodiesterase, suggesting that the delta subunit bound preferentially to the methylated enzyme in the outer segment. Together these results suggest that the delta subunit/phosphodiesterase catalytic subunit interaction may be regulated by the C-terminal methylation of the catalytic subunits.

Characterization of human and mouse rod cGMP phosphodiesterase delta subunit (PDE6D) and chromosomal localization of the human gene.

The mammalian multisubunit photoreceptor cGMP phosphodiesterase PDE alpha beta gamma 2 (PDE6 family) is a peripherally membrane-associated enzyme. A novel subunit, termed PDE delta (HGMW-approved symbol, PDE6D; MW 17 kDa), is able to detach PDE partially from bovine rod outer segment membranes under physiological conditions. Cloning of human and mouse PDE delta cDNAs revealed that PDE delta is a nearly perfectly conserved polypeptide of 150 amino acids that shows partial sequence homology to photoreceptor RG4 of unknown function. Multiple-species Southern blot analysis demonstrates that the PDE delta gene has been well conserved during evolution and is detectable at high stringency in invertebrates. The human and mouse genes are contained in less than 8 kb of genomic DNA and consist of four exons and three introns (0.7-4 kb in human, 0.7-2.2 kb in mouse). The PDE delta gene structure is identical to that of the C27H5.1 gene identified in the eyeless nematode Caenorhabditis elegans. The human PDE delta gene (locus designation PDE6D) was localized to the long arm of chromosome 2 (2q35-q36) by fluorescence in situ hybridization. By synteny, the mouse PDE delta gene is predicted to reside on chromosome 1.

Stimulation of p85/RING3 kinase in multiple organs after systemic administration of mitogens into mice.

Using an autophosphorylation membrane assay, we examined activation of kinases in different organs after intraperitoneal injections of mitogens and cytokines into mice. In the multiple organs examined administration of either epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA) or interleukin-1beta (IL-1beta) activated a number of kinases. Most notably among those was a kinase of approximately 85 kDa (p85) that was activated by EGF, PMA and IL-1beta in the lung, kidney, brain, liver and heart. The size and properties of this enzyme are indistinguishable from the RING3 kinase that has a very high activity in leukocytes of patients with leukemia. In animals treated with PMA, antibodies against RING3 kinase immunoprecipitated PMA-responsive p85 activity from the lung and brain suggesting that p85 and RING3 kinases are the same enzymes. Activation of p85/RING3 kinase by growth factors in multiple organs might reflect involvement of this enzyme in the pathogenesis of leukemias and other proliferative diseases.

Solubilization of membrane-bound rod phosphodiesterase by the rod phosphodiesterase recombinant delta subunit.

Retinal rod and cone phosphodiesterases are oligomeric enzymes that consist of a dimeric catalytic core (alpha'2 in cones and alphabeta in rods) with inhibitory subunits (gamma) that regulate their activity. In addition, a 17-kDa protein referred to as the delta subunit co-purifies with the rod soluble phosphodiesterase and the cone phosphodiesterase. We report here partial protein sequencing of the rod delta subunit and isolation of a cDNA clone encoding it. The predicted amino acid sequence is unrelated to any other known protein. Of eight bovine tissue mRNA preparations examined by Northern analysis, the strongest delta subunit-specific signal was present in the retina. A less intense signal was seen in the brain and adrenal mRNA. In bovine retinal sections, rod delta subunit anti-peptide antibodies label rod but not cone outer segments. delta subunit, added back to washed outer segment membranes, solubilizes a large fraction of the membrane-bound phosphodiesterase, indicating that this subunit binds to the classical membrane associated phosphodiesterase. The subunit forms a tight complex with native, but not trypsin-released phosphodiesterase, suggesting that the isoprenylated carboxyl termini of the catalytic subunits may be involved in binding of the delta subunit to the phosphodiesterase holoenzyme.

Membrane skeleton protein 4.1 in developing Xenopus: expression in postmitotic cells of the retina.

Membrane skeleton protein 4.1 plays a key role in modulating the interactions of spectrin, actin, and integral membrane proteins in erythroid and nonerythroid cells. We have investigated its structure and expression during embryonic development of Xenopus laevis. An analysis of the complete 2758-nucleotide sequence and predicted translation of 801 amino acids (85.5 kDa) of X. laevis oocyte protein 4.1 reveals that, within overlapping regions, oocyte protein 4.1 is 74% identical to a composite amino acid sequence of human erythroid and lymphoid protein 4.1 and has an identity similar to that of amino acid motifs variably expressed in either human erythroid or lymphoid protein 4.1 S1 nuclease protection analysis demonstrates the presence of a single species of protein 4.1 transcript in embryos. Antibodies produced against X. laevis protein 4.1 fusion protein recognize two bands of 180 and 115 kDa on Western blots of X. laevis embryos and retina and, using immunocytochemical techniques, label the developing retina most intensely. In vitro transcription of a cDNA construct fully encoding X. laevis protein 4.1 yields a synthetic mRNA which, when translated in vitro, produces a polypeptide that comigrates on SDS-polyacrylamide gels with the 115-kDa form of embryos and retina. Protein 4.1 is found exclusively in photoreceptors following the terminal mitosis of retinal neurons. When retinal synaptogenesis is complete, protein 4.1 is also expressed in the inner retina. In adult amphibian retinas, protein 4.1 is detected in photoreceptors, bipolar cells, and ganglion cell axons. As these cell types have previously been shown to express spectrin, actin, and ankyrin, it is likely that the membrane skeleton of erythrocytes and retinal cells share functional similarities.

Transient potassium currents in avian sensory neurons.

1. We used the patch-clamp technique to study voltage-activated transient potassium currents in freshly dispersed and cultured chick dorsal root ganglion (DRG) cells. Whole-cell and cell-attached patch currents were recorded under conditions appropriate for recording potassium currents. 2. In whole-cell experiments, 100-ms depolarizations from normal resting potentials (-50 to -70 mV) elicited sustained outward currents that inactivated over a time scale of seconds. We attribute this behavior to a component of delayed rectifier current. After conditioning hyperpolarizations to potentials negative to -80 mV, depolarizations elicited transient outward current components that inactivated with time constants in the range of 8-26 ms. We attribute this behavior to a transient outward current component. 3. Conditioning hyperpolarizations increased the rate of activation of the net outward current implying that the removal of inactivation of the transient outward current allows it to contribute to early outward current during depolarizations from negative potentials. 4. Transient current was more prominent on the day the cells were dispersed and decreased with time in culture. 5. In cell-attached patches, single channels mediating outward currents were observed that were inactive at resting potentials but were active transiently during depolarizations to potentials positive to -30 mV. The probability of channels being open increased rapidly (peaking within approximately 6 ms) and then declined with a time constant in the range of 13-30 ms. With sodium as the main extracellular cation, single-channel conductances ranged from 18 to 32 pS. With potassium as the main extracellular cation, the single-channel conductance was approximately 43 pS, and the channel current reversed near 0 mV, as expected for a potassium current. 6. We conclude that the transient potassium channels mediate the component of transient outward current seen in the whole-cell experiments. This current is a relatively small component of the net current during depolarizations from normal resting potentials, but it can contribute significant outward current early in depolarizations from hyperpolarized potentials.