Mariangela Chisari

Shuttling of G Protein Subunits between the Plasma Membrane and Intracellular Membranes

Heterotrimeric G proteins (αβγ) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t½ < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

The sticky issue of neurosteroids and GABAA receptors

Endogenous neurosteroids and their synthetic analogs (neuroactive steroids) are potent modulators of GABA(A) receptors. Thus, they are of physiological and clinical relevance for their ability to modulate inhibitory function in the CNS. Despite their importance, fundamental issues of neurosteroid actions remain unresolved. Recent evidence suggests that glutamatergic principal neurons, rather than glia, are the major sources of neurosteroid synthesis. Other recent studies have identified putative neurosteroid binding sites on GABA(A) receptors. In this Opinion, we argue that neurosteroids require a membranous route of access to transmembrane-domain binding sites within GABA(A) receptors. This has implications for the design of future neuroactive steroids because the lipid solubility and related accessibility properties of the ligand are likely to be key determinants of receptor modulation.

A family of G protein βγ subunits translocate reversibly from the plasma membrane to endomembranes on receptor activation.

The present model of G protein activation by G protein-coupled receptors exclusively localizes their activation and function to the plasma membrane (PM). Observation of the spatiotemporal response of G protein subunits in a living cell to receptor activation showed that 6 of the 12 members of the G protein γ subunit family translocate specifically from the PM to endomembranes. The γ subunits translocate as βγ complexes, whereas the α subunit is retained on the PM. Depending on the γ subunit, translocation occurs predominantly to the Golgi complex or the endoplasmic reticulum. The rate of translocation also varies with the γ subunit type. Different γ subunits, thus, confer distinct spatiotemporal properties to translocation. A striking relationship exists between the amino acid sequences of various γ subunits and their translocation properties. γ subunits with similar translocation properties are more closely related to each other. Consistent with this relationship, introducing residues conserved in translocating subunits into a non-translocating subunit results in a gain of function. Inhibitors of vesicle-mediated trafficking and palmitoylation suggest that translocation is diffusion-mediated and controlled by acylation similar to the shuttling of G protein subunits (Chisari, M., Saini, D. K., Kalyanaraman, V., and Gautam, N. (2007) J. Biol. Chem. 282, 24092–24098). These results suggest that the continual testing of cytosolic surfaces of cell membranes by G protein subunits facilitates an activated cell surface receptor to direct potentially active G protein βγ subunits to intracellular membranes.

Diverse voltage-sensitive dyes modulate GABAA receptor function

Voltage-sensitive dyes are important tools for assessing network and single-cell excitability, but an untested premise in most cases is that the dyes do not interfere with the parameters (membrane potential, excitability) that they are designed to measure. We found that popular members of several different families of voltage-sensitive dyes modulate GABAA receptor with maximum efficacy and potency similar to clinically used GABAA receptor modulators. Di-4-ANEPPS and DiBAC4(3) potentiated GABA function with micromolar and high nanomolar potency, respectively, and yielded strong maximum effects similar to barbiturates and neurosteroids. Newer blue oxonols had biphasic effects on GABAA receptor function at nanomolar and micromolar concentrations, with maximum potentiation comparable to that of saturating benzodiazepine effects. ANNINE-6 and ANNINE-6plus had no detectable effect on GABAA receptor function. Even dyes with no activity on GABAA receptors at baseline induced photodynamic enhancement of GABAA receptors. The basal effects of dyes were sufficient to prolong IPSCs and to dampen network activity in multielectrode array recordings. Therefore, the dual effects of voltage-sensitive dyes on GABAergic inhibition require caution in dye use for studies of excitability and network activity.

Erratic expression of DNA polymerases by β-amyloid causes neuronal death

An ectopic reentrance into the cell cycle with ensuing DNA replication is required for neuronal apoptosis induced by β‐amyloid. Here, we investigate the repertoire of DNA polymerases expressed in β‐amyloid‐treated neurons, and their specific role in DNA synthesis and apoptosis. We show that exposure of cultured cortical neurons to β‐amyloid induces the expression of DNA polymerase‐β, proliferating cell nuclear antigen, and the p49 and p58 subunits of DNA primase. Induction requires the activity of cyclin‐dependent kinases. The knockdown of the p49 primase subunit prevents β‐amyloid‐induced neuronal DNA synthesis and apoptosis. Similar effects are observed by knocking down DNA polymerase‐β or by using dideoxycytidine, a preferential inhibitor of this enzyme. Thus, the reparative enzyme DNA polymerase‐β unexpectedly mediates a large component of de novo DNA synthesis and apoptotic death in neurons exposed to βamyloid. These data indicate that DNA polymerases become death signals when erratically expressed by differentiated neurons.

The Influence of Neuroactive Steroid Lipophilicity on GABAA Receptor Modulation: Evidence for a Low-Affinity Interaction

Anesthetic steroids with actions at gamma-aminobutyric acid type A receptors (GABA(A)Rs) may access transmembrane domain binding site(s) directly from the plasma cell membrane. Accordingly, the effective concentration in lipid phase and the ability of the steroid to meet pharmacophore requirements for activity will both contribute to observed steady-state potency. Furthermore, onset and offset of receptor effects may be rate limited by lipid partitioning. Here we show that several GABA-active steroids, including naturally occurring neurosteroids, of different lipophilicity differ in kinetics and potency at GABA(A)Rs. The hydrophobicity ranking predicted relative potency of GABA(A)R potentiation and predicted current offset kinetics. Kinetic offset differences among steroids were largely eliminated by gamma-cyclodextrin, a scavenger of unbound steroid, suggesting that affinity differences among the analogues are dwarfed by the contributions of nonspecific accumulation. A 7-nitrobenz-2-oxa-1,3-diazole (NBD)-tagged fluorescent analogue of the low-lipophilicity alphaxalone (C17-NBD-alphaxalone) exhibited faster nonspecific accumulation and departitioning than those of a fluorescent analogue of the high-lipophilicity (3alpha,5alpha)-3-hydroxypregnan-20-one (C17-NBD-3alpha5alphaA). These differences were paralleled by differences in potentiation of GABA(A)R function. The enantiomer of C17-NBD-3alpha5alphaA, which does not satisfy pharmacophore requirements for steroid potentiation, exhibited identical fluorescence kinetics and distribution to C17-NBD-3alpha5alphaA, but was inactive at GABA(A)Rs. Simple simulations supported our major findings, which suggest that neurosteroid binding affinity is low. Therefore both specific (e.g., fulfilling pharmacophore requirements) and nonspecific (e.g., lipid solubility) properties contribute to the potency and longevity of anesthetic steroid action.

Shuttling and translocation of heterotrimeric G proteins and Ras

Heterotrimeric G proteins (alphabetagamma) and Ras proteins are activated by cell-surface receptors that sense extracellular signals. Both sets of proteins were traditionally thought to be constrained to the plasma membrane and some intracellular membranes. Live-cell-imaging experiments have now shown that these proteins are mobile inside a cell, shuttling continually between the plasma membrane and intracellular membranes in the basal state, maintaining these proteins in dynamic equilibrium in different membrane compartments. Furthermore, on receptor activation, a family of G protein betagamma subunits translocates rapidly and reversibly to the Golgi and endoplasmic reticulum enabling direct communication between the extracellular signal and intracellular membranes. A member of the Ras family has similarly been shown to translocate on activation. Although the impact of this unexpected intracellular movement of signaling proteins on cell physiology is likely to be distinct, there are striking similarities in the properties of these two families of signal-transducing proteins.

A Mechanism Regulating G Protein-coupled Receptor Signaling That Requires Cycles of Protein Palmitoylation and Depalmitoylation

Background: The functions of palmitate turnover in signal transduction are poorly understood. Results: Inhibiting palmitate turnover on R7BP redistributed R7BP-R7 RGS complexes from the plasma membrane to endomembranes, dissociated them from GIRK channels, and delayed Gi/o deactivation and channel closure. Conclusion: Palmitate turnover on R7BP promotes GIRK channel deactivation. Significance: Inhibiting palmitate turnover on R7BP could enhance GIRK activity in neurological disorders. Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K+ (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.

GIRK channel modulation by assembly with allosterically regulated RGS proteins

G-protein–activated inward-rectifying K+ (GIRK) channels hyperpolarize neurons to inhibit synaptic transmission throughout the nervous system. By accelerating G-protein deactivation kinetics, the regulator of G-protein signaling (RGS) protein family modulates the timing of GIRK activity. Despite many investigations, whether RGS proteins modulate GIRK activity in neurons by mechanisms involving kinetic coupling, collision coupling, or macromolecular complex formation has remained unknown. Here we show that GIRK modulation occurs by channel assembly with R7-RGS/Gβ5 complexes under allosteric control of R7 RGS-binding protein (R7BP). Elimination of R7BP occludes the Gβ5 subunit that interacts with GIRK channels. R7BP-bound R7-RGS/Gβ5 complexes and Gβγ dimers interact noncompetitively with the intracellular domain of GIRK channels to facilitate rapid activation and deactivation of GIRK currents. By disrupting this allosterically regulated assembly mechanism, R7BP ablation augments GIRK activity. This enhanced GIRK activity increases the drug effects of agonists acting at G-protein–coupled receptors that signal via GIRK channels, as indicated by greater antinociceptive effects of GABA(B) or μ-opioid receptor agonists. These findings show that GIRK current modulation in vivo requires channel assembly with allosterically regulated RGS protein complexes, which provide a target for modulating GIRK activity in neurological disorders in which these channels have crucial roles, including pain, epilepsy, Parkinson’s disease and Down syndrome.

Long-term incubation with β-amyloid peptides impairs endothelium-dependent vasodilatation in isolated rat basilar artery

Alzheimers disease is associated to a cerebral amyloid angiopathy with dysregulation of cerebral blood flow (CBF). In vitro studies have shown that short-term application of beta-amyloid (Abeta) peptides to isolated vessels affects vascular tone within 1h, but no studies have examined the effect of long-term incubation with Abeta. Here we evaluate the effect of Abeta((1-40)) and Abeta((25-35)) in rat basilar artery for up to 24h. Basilar artery segments were incubated with 25microeta((1-40)) or Abeta((25-35)), for 6 or 24h. After treatment, arteries were mounted in a wire myograph, in physiological salt solution gassed with O(2)/CO(2), in the absence of Abeta, and challenged with vasoconstrictors and vasodilators. Vasomotor responses were not significantly changed by 6h treatment with Abeta peptides whereas 24h treatment with either Abeta((25-35)) or Abeta((1-40)) increased vasoconstriction to 5-hydroxytryptamine (5-HT) and reduced endothelium-dependent vasodilatation to acetylcholine (ACh). Analysis of endothelial cells did not show apoptotic changes associated to endothelial dysfunction, as assessed by TUNEL immunostaining and examination of nuclear morphology, but basal phosphorylation of endothelial nitric oxide synthase (at serine 1177) appeared reduced. These data suggest that long incubation with Abeta peptides induces an alteration of endothelial function in isolated basilar artery, involving eNOS activity without changing cell morphology. This endothelial dysfunction may play a role in the pathogenesis of CBF dysregulation occurring in cerebral amyloid angiopathy and Alzheimers disease.