RGS4 and GAIP are GTPase-activating proteins for Gqα and block activation of phospholipase Cβ by γ-thio-GTP-Gqα

RGS proteins constitute a newly appreciated and large group of negative regulators of G protein signaling. Four members of the RGS family act as GTPase-activating proteins (GAPs) with apparent specificity for members of the G_iα subfamily of G protein subunits. We demonstrate here that two RGS proteins, RGS4 and GAIP, also act as GAPs for G_qα, the G_α protein responsible for activation of phospholipase Cβ. Furthermore, these RGS proteins block activation of phospholipase Cβ by guanosine 5′-(3-O-thio)triphosphate-G_qα. GAP activity does not explain this effect, which apparently results from occlusion of the binding site on G_α for effector. Inhibitory effects of RGS proteins on G protein-mediated signaling pathways can be demonstrated by simple mixture of RGS4 or GAIP with plasma membranes.     

Fidelity of G protein β-subunit association by the G protein γ-subunit-like domains of RGS6, RGS7, and RGS11

Several regulators of G protein signaling (RGS) proteins contain a G protein gamma-subunit-like (GGL) domain, which, as we have shown, binds to Gbeta5 subunits. Here, we extend our original findings by describing another GGL-domain-containing RGS, human RGS6. When RGS6 is coexpressed with different Gbeta subunits, only RGS6 and Gbeta5 interact. The expression of mRNA for RGS6 and Gbeta5 in human tissues overlaps. Predictions of alpha-helical and coiled-coil character within GGL domains, coupled with measurements of Gbeta binding by GGL domain mutants, support the contention that Ggamma-like regions within RGS proteins interact with Gbeta5 subunits in a fashion comparable to conventional Gbeta/Ggamma pairings. Mutation of the highly conserved Phe-61 residue of Ggamma2 to tryptophan, the residue present in all GGL domains, increases the stability of the Gbeta5/Ggamma2 heterodimer, highlighting the importance of this residue to GGL/Gbeta5 association.     

Inhibition of regulator of G protein signaling function by two mutant RGS4 proteins

Regulators of G protein signaling (RGS) proteins limit the lifetime of activated (GTP-bound) heterotrimeric G protein α subunits by acting as GTPase-activating proteins (GAPs). Mutation of two residues in RGS4, which, based on the crystal structure of RGS4 complexed with G_iα1-GDP-AlF₄⁻, directly contact G_iα1 (N88 and L159), essentially abolished RGS4 binding and GAP activity. Mutation of another contact residue (S164) partially inhibited both binding and GAP activity. Two other mutations, one of a contact residue (R167M/A) and the other an adjacent residue (F168A), also significantly reduced RGS4 binding to G_iα1-GDP-AlF₄⁻, but in addition redirected RGS4 binding toward the GTPγS-bound form. These two mutant proteins had severely impaired GAP activity, but in contrast to the others behaved as RGS antagonists in GAP and in vivo signaling assays. Overall, these results are consistent with the hypothesis that the predominant role of RGS proteins is to stabilize the transition state for GTP hydrolysis. In addition, mutant RGS proteins can be created with an altered binding preference for the G_iα-GTP conformation, suggesting that efficient RGS antagonists can be developed.     

Attenuation of Gi- and Gq-mediated signaling by expression of RGS4 or GAIP in mammalian cells

Protein regulators of G protein signaling (RGS proteins) were discovered as negative regulators of heterotrimeric G protein-mediated signal transduction in yeast and worms. Experiments with purified recombinant proteins in vitro have established that RGS proteins accelerate the GTPase activity of certain G protein α subunits (the reaction responsible for their deactivation); they can also act as effector antagonists. We demonstrate herein that either of two such RGS proteins, RGS4 or GAIP, attenuated signal transduction mediated by endogenous receptors, G proteins, and effectors when stably expressed as tagged proteins in transfected mammalian cells. The pattern of selectivity observed in vivo was similar to that seen in vitro. RGS4 and GAIP both attenuated G_i-mediated inhibition of cAMP synthesis. RGS4 was more effective than GAIP in blocking G_q-mediated activation of phospholipase Cβ.     

RGS2/G0S8 is a selective inhibitor of Gqα function

RGS (regulators of G protein signaling) proteins are GTPase activating proteins that inhibit signaling by heterotrimeric G proteins. All RGS proteins studied to date act on members of the Giα family, but not Gsα or G12α. RGS4 regulates Giα family members and Gqα. RGS2 (G0S8) is exceptional because the G proteins it regulates have not been identified. We report that RGS2 is a selective and potent inhibitor of Gqα function. RGS2 selectively binds Gqα, but not other Gα proteins (Gi, Go, Gs, G12/13) in brain membranes; RGS4 binds Gqα and Giα family members. RGS2 binds purified recombinant Gqα, but not Goα, whereas RGS4 binds either. RGS2 does not stimulate the GTPase activities of Gsα or Giα family members, even at a protein concentration 3000-fold higher than is sufficient to observe effects of RGS4 on Giα family members. In contrast, RGS2 and RGS4 completely inhibit Gq-directed activation of phospholipase C in cell membranes. When reconstituted with phospholipid vesicles, RGS2 is 10-fold more potent than RGS4 in blocking Gqα-directed activation of phospholipase Cβ1. These results identify a clear physiological role for RGS2, and describe the first example of an RGS protein that is a selective inhibitor of Gqα function.     

The core domain of a new retina specific RGS protein stimulates the GTPase activity of transducin in vitro

GTP hydrolysis by the transducin α subunit is stimulated by a membrane-bound protein. The identity of this GTPase-activating protein (GAP) is not yet known, but the recent identification of a new gene family encoding regulator of G protein signaling (RGS) proteins raises the possibility that the transducin GAP is an RGS protein. Biochemical evidence shows that RGS proteins act as GAPs for α subunits of the G_i subfamily of G proteins. To identify an RGS protein that could be a GAP for the transducin α subunit, we investigated the expression of RGS proteins in the retina and identified a new RGS domain, RET-RGS-d, which is specifically expressed in the retina. In situ RNA hybridization analyses revealed that RET-RGS-d is expressed in photoreceptor cells as well as in other cells of the retina. Recombinant RET-RGS-d accelerates single turnover hydrolysis of GTP by transducin. We used RET-RGS-d to isolate a full-length cDNA, RET-RGS1, encoding a new RGS protein with a C terminus that corresponds to RET-RGS-d. The N-terminal half of RET-RGS1 contains a putative transmembrane domain and a string of nine cysteines that are potential substrates for multiple palmitoylation. These findings suggest that RET-RGS1 is an integral membrane protein and that it is a candidate for the membrane-associated protein responsible for the GAP activity detected in photoreceptor membranes.     

Targeted inactivation of αi2 or αi3 disrupts activation of the cardiac muscarinic K+ channel, IK+Ach, in intact cells

Cardiac muscarinic receptors activate an inwardly rectifying K⁺ channel, I_K^+Ach, via pertussis toxin (PT)-sensitive heterotrimeric G proteins (in heart G_i2, G_i3, or G_o). We have used embryonic stem cell (ES cell)-derived cardiocytes with targeted inactivations of specific PT-sensitive α subunits to determine which G proteins are required for receptor-mediated regulation of I_K^+Ach in intact cells. The muscarinic agonist carbachol increased I_K^+Ach activity in ES cell-derived cardiocytes from wild-type cells, in cells lacking α_o, and in cells lacking the PT-insensitive G protein α_q. In cells with targeted inactivation of α_i2 or α_i3, channel activation by both carbachol and adenosine was blocked. Carbachol-induced channel activation was restored in the α_i2- and α_i3-null cells by reexpressing the previously targeted gene and guanosine 5′-[γ-thio] triphosphate was able to fully activate I_K^+Ach in excised membranes patches from these mutants. In contrast, negative chronotropic responses to both carbachol and adenosine were preserved in cells lacking α_i2 or α_i3. Our results show that expression of two specific PT-sensitive α subunits (α_i2 and α_i3 but not α_o) is required for normal agonist-dependent activation of I_K^+Ach and suggest that both α_i2- and α_i3-containing heterotrimeric G proteins may be involved in the signaling process. Also the generation of negative chronotropic responses to muscarinic or adenosine receptor agonists do not require activation of I_K^+Ach or the expression of α_i2 or α_i3.     

Activation of the atrial KACh channel by the βγ subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidylinositol phosphates

The βγ subunits of GTP-binding proteins (G_βγ) activate the muscarinic K⁺ channel (K_ACh) in heart by direct binding to both of its component subunits. K_ACh channels can also be gated by internal Na⁺ ions. Both activation mechanisms show dependence on hydrolysis of intracellular ATP. We report that phosphatidylinositol 4,5-bisphosphate (PIP₂) mimics the ATP effects and that depletion or block of PIP₂ retards the stimulatory effects of G_βγ subunits or Na⁺ ions on channel activity, effects that can be reversed by restoring PIP₂. Thus, regulation of K_ACh channel activity may be crucially dependent on PIP₂ and phosphatidylinositol signaling. These striking functional results are in agreement with in vitro biochemical studies on the PIP₂ requirement for G_βγ stimulation of G protein receptor kinase activity, thus implicating phosphatidylinositol phospholipids as a potential control point for G_βγ-mediated signal transduction.     

RGS proteins reconstitute the rapid gating kinetics of Gβγ-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K⁺ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Gα(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling” (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m₂ receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.     

Chemotaxis in a lymphocyte cell line transfected with C-C chemokine receptor 2B: Evidence that directed migration is mediated by βγ dimers released by activation of Gαi-coupled receptors

Chemotaxis is mediated by activation of seven-transmembrane domain, G protein-coupled receptors, but the signal transduction pathways leading to chemotaxis are poorly understood. To identify G proteins that signal the directed migration of cells, we stably transfected a lymphocyte cell line (300-19) with G protein-coupled receptors that couple exclusively to G_αq (the m3 muscarinic receptor), G_αi (the κ-opioid receptor), and G_αs (the β-adrenergic receptor), as well as the human thrombin receptor (PAR-1) and the C-C chemokine receptor 2B. Cells expressing receptors that coupled to G_αi, but not to G_αq or G_αs, migrated in response to a concentration gradient of the appropriate agonist. Overexpression of G_α transducin, which binds to and inactivates free G_βγ dimers, completely blocked chemotaxis although having little or no effect on intracellular calcium mobilization or other measures of cell signaling. The identification of G_βγ dimers as a crucial intermediate in the chemotaxis signaling pathway provides further evidence that chemotaxis of mammalian cells has important similarities to polarized responses in yeast. We conclude that chemotaxis is dependent on activation of G_αi and the release of G_βγ dimers, and that G_αi-coupled receptors not traditionally associated with chemotaxis can mediate directed migration when they are expressed in hematopoietic cells.     

Evidence that the nucleotide exchange and hydrolysis cycle of G proteins causes acute desensitization of G-protein gated inward rectifier K+ channels

The G-protein gated inward rectifier K⁺ channel (GIRK) is activated in vivo by the Gβγ subunits liberated upon G_i-coupled receptor activation. We have recapitulated the acute desensitization of receptor-activated GIRK currents in heterologous systems and shown that it is a membrane-delimited process. Its kinetics depends on the guanine nucleotide species available and could be accounted for by the nucleotide exchange and hydrolysis cycle of G proteins. Indeed, acute desensitization is abolished by nonhydrolyzable GTP analogues. Whereas regulators of G-protein signaling (RGS) proteins by their GTPase-activating protein activities are regarded as negative regulators, a positive regulatory function of RGS4 is uncovered in our study; the opposing effects allow RGS4 to potentiate acute desensitization without compromising GIRK activation.     

Gsα-selective G protein antagonists

Suramin acts as a G protein inhibitor because it inhibits the rate-limiting step in activation of the G_α subunit, i.e., the exchange of GDP for GTP. Here, we have searched for analogues that are selective for G_sα. Two compounds have been identified: NF449 (4,4′,4",4′"-[carbonyl-bis[imino-5,1,3-benzenetriyl bis-(carbonylimino)]]tetrakis-(benzene-1,3-disulfonate) and NF503 (4,4′-[carbonylbis[imino-3,1-phenylene-(2,5-benzimidazolylene)carbonylimino]]bis-benzenesulfonate). These compounds (i) suppress the association rate of guanosine 5′-[γ-thio]triphosphate ([³⁵S]GTP[γS]) binding to G_sα-s but not to G_iα-1, (ii) inhibit stimulation of adenylyl cyclase activity in S49 cyc⁻ membranes (deficient in endogenous G_sα) by exogenously added G_sα-s, and (iii) block the coupling of β-adrenergic receptors to G_s with half-maximum effects in the low micromolar range. In contrast to suramin, which is not selective, NF503 and NF449 disrupt the interaction of the A₁-adenosine receptor with its cognate G proteins (G_i/G_o) at concentrations that are >30-fold higher than those required for uncoupling of β-adrenergic receptor/G_s tandems; similarly, the angiotensin II type-1 receptor (a prototypical G_q-coupled receptor) is barely affected by the compounds. Thus, NF503 and NF449 fulfill essential criteria for G_sα-selective antagonists. The observations demonstrate the feasibility of subtype-selective G protein inhibition.     

Decay of prepulse facilitation of N type calcium channels during G protein inhibition is consistent with binding of a single Gβγ subunit

We have examined the modulation of cloned and stably expressed rat brain N type calcium channels (α_1B + β_1b + α₂δ subunits) by exogenously applied purified G protein βγ subunits. In the absence of G_βγ, barium currents through N type channels are unaffected by application of strong depolarizing prepulses. In contrast, inclusion of purified G_βγ in the patch pipette results in N type currents that initially facilitated upon application of positive prepulses followed by rapid reinhibition. Examination of the kinetics of G_βγ-dependent reinhibition showed that as the duration between the test pulse and the prepulse was increased, the degree of facilitation was attenuated in a monoexponential fashion. The time constant τ for the recovery from facilitation was sensitive to exogenous G_βγ, so that the inverse of τ linearly depended on the G_βγ concentration. Overall, the data are consistent with a model whereby a single G_βγ molecule dissociates from the channel during the prepulse, and that reassociation of G_βγ with the channel after the prepulse occurs as a bimolecular reaction.     

Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel α1A subunit

Synaptic transmission is regulated by G protein-coupled receptors whose activation releases G protein βγ subunits that modulate presynaptic Ca²⁺ channels. The sequence motif QXXER has been proposed to be involved in the interaction between G protein βγ subunits and target proteins including adenylyl cyclase 2. This motif is present in the intracellular loop connecting domains I and II (L_I-II) of Ca²⁺ channel α_1A subunits, which are modulated by G proteins, but not in α_1C subunits, which are not modulated. Peptides containing the QXXER motif from adenylate cyclase 2 or from α_1A block G protein modulation but a mutant peptide containing the sequence AXXAA does not, suggesting that the QXXER-containing peptide from α_1A can competitively inhibit Gβγ modulation. Conversion of the R in the QQIER sequence of α_1A to E as in α_1C slows channel inactivation and shifts the voltage dependence of steady-state inactivation to more positive membrane potentials. Conversion of the final E in the QQLEE sequence of α_1C to R has opposite effects on voltage-dependent inactivation, although the changes are not as large as those for α_1A. Mutation of the QQIER sequence in α_1A to QQIEE enhanced G protein modulation, and mutation to QQLEE as in α_1C greatly reduced G protein modulation and increased the rate of reversal of G protein effects. These results indicate that the QXXER motif in L_I-II is an important determinant of both voltage-dependent inactivation and G protein modulation, and that the amino acid in the third position of this motif has an unexpectedly large influence on modulation by Gβγ. Overlap of this motif with the consensus sequence for binding of Ca²⁺ channel β subunits suggests that this region of L_I-II is important for three different modulatory influences on Ca²⁺ channel activity.     

Instability of GGL domain-containing RGS proteins in mice lacking the G protein beta-subunit Gbeta5

RGS (regulator of G protein signaling) proteins containing the G protein gamma-like (GGL) domain (RGS6, RGS7, RGS9, and RGS11) interact with the fifth member of the G protein beta-subunit family, Gbeta5. This interaction is necessary for the stability of both the RGS protein and for Gbeta5. Consistent with this notion, we have found that elevation of RGS9-1 mRNA levels by transgene expression does not increase RGS9-1 protein level in the retina, suggesting that Gbeta5 levels may be limiting. To examine further the interactions of Gbeta5 and the GGL domain-containing RGS proteins, we inactivated the Gbeta5 gene. We found that the levels of GGL domain-containing RGS proteins in retinas and in striatum are eliminated or reduced drastically, whereas the levels of Ggamma2 and RGS4 proteins remain normal in the absence of Gbeta5. The homozygous Gbeta5 knockout (Gbeta5-/-) mice derived from heterozygous knockout mating are runty and exhibit a high preweaning mortality rate. We concluded that complex formation between GGL domain-containing RGS proteins and the Gbeta5 protein is necessary to maintain their mutual stability in vivo. Furthermore, in the absence of Gbeta5 and all four RGS proteins that form protein complexes with Gbeta5, the animals that survive into adulthood are viable and have no gross defects in brain or retinal morphology.     

Identification of components of a phosphoinositide signaling pathway in retinal rod outer segments

Phototransduction in retinal rods involves a G protein-coupled signaling cascade that leads to cGMP hydrolysis and the closure of cGMP-gated cation channels that are open in darkness, producing a membrane hyperpolarization as the light response. For many years there have also been reports of the presence of a phosphoinositide pathway in the rod outer segment, though its functions and the molecular identities of its components are still unclear. Using immunocytochemistry with antibodies against various phosphoinositide-specific phospholipase C (PLC) isozymes (β1–4, γ1–2, and δ1–2), we have found PLCβ4-like immunoreactivity in rod outer segments. Similar experiments with antibodies against the α-subunits of the G_q family of G proteins, which are known to activate PLCβ4, have also demonstrated G_α11-like immunoreactivity in this location. Immunoblots of total proteins from whole retina or partially purified rod outer segments with anti-PLCβ4 and anti-G_α11 antibodies gave, respectively, a single protein band of the expected molecular mass, suggesting specific labelings. The retinal locations of the two proteins were also supported by in situ hybridization experiments on mouse retina with probes specific for the corresponding mouse genes. These two proteins, or immunologically identical isoforms, therefore likely mediate the phosphoinositide signaling pathway in the rod outer segment. At present, G_α11 or a G_α11-like protein represents the only G protein besides transducin (which mediates phototransduction) identified so far in the rod outer segment. Although absent in the outer segment layer, other PLC isoforms as well as G_αq (another G_q family member), are present elsewhere in the retina.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Folding of the N-terminal, ligand-binding region of integrin α-subunits into a β-propeller domain

The N-terminal ≈440 aa of integrin α subunits contain seven sequence repeats. These are predicted here to fold into a β-propeller domain. A homologous domain from the enzyme phosphatidylinositol phospholipase D is predicted to have the same fold. The domains contain seven four-stranded β-sheets arranged in a torus around a pseudosymmetry axis. The trimeric G-protein β subunit (G beta) appears to be the most closely related β-propeller. Integrin ligands and a putative Mg²⁺ ion are predicted to bind to the upper face of the β-propeller. This face binds substrates in β-propeller enzymes and is used by the G protein β subunit to bind the G protein α subunit. The integrin α subunit I domain, which is structurally homologous to the G protein α subunit, is tethered to the top of the β-propeller domain by a hinge that may allow movement of the domains relative to one another. The Ca²⁺-binding motifs in integrin α subunits are on the lower face of the β-propeller.     

Gsα contains an unidentified covalent modification that increases its affinity for adenylyl cyclase

Many G protein α subunits are dually acylated with myristate and palmitate or are palmitoylated on more than one cysteine residue near their N termini. The G_α protein that activates adenylyl cyclase, α_s, is not myristoylated but can be reversibly palmitoylated. It appears that α_s contains another, as-yet-unidentified covalent modification that decreases its apparent dissociation constant for adenylyl cyclase from 50 nM to <0.5 nM. This modification is at or near the N terminus of the protein and is hydrophobic. Palmitoylation of native α_s does not account for its high affinity for adenylyl cyclase.     

Presynaptic α2δ subunits are key organizers of glutamatergic synapses

In nerve cells the genes encoding for α₂δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α₂δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α₂δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α₂δ isoforms as synaptic organizers is highly redundant, as each individual α₂δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α₂δ-2 and α₂δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α₂δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α₂δ implicates α₂δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α₂δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.

In synapses neurotransmitter release is triggered by the entry of calcium through voltage-gated calcium channels (VGCCs). Neuronal VGCCs consist of an ion-conducting α₁ subunit and the auxiliary β and α₂δ subunits. α₂δ subunits, the targets of the widely prescribed antiepileptic and antiallodynic drugs gabapentin and pregabalin, are membrane-anchored extracellular glycoproteins, which modulate VGCC trafficking and calcium currents (1–5). In nerve cells α₂δ subunits have been linked to neuropathic pain and epilepsy (4) and they interact with mutant prion proteins (6) and regulate synaptic release probability (7). Importantly, all α₂δ isoforms are implicated in synaptic functions. Presynaptic effects of α₂δ-1, for example, may be mediated by an interaction with α-neurexins (8) or N-methyl-D-aspartate receptors (e.g., refs. 9 and 10). In contrast, postsynaptic α₂δ-1 acts as a receptor for thrombospondins (11) and promotes spinogenesis via postsynaptic Rac1 (12). α₂δ-2 is necessary for normal structure and function of auditory hair cell synapses (13); it has been identified as a regulator of axon growth and hence a suppressor of axonal regeneration (14) and was recently shown to control structure and function of cerebellar climbing fiber synapses (15). A splice variant of α₂δ-2 regulates postsynaptic GABA_A receptor (GABA_AR) abundance and axonal wiring (16). In invertebrates, α₂δ loss of function was associated with abnormal presynaptic development in motoneurons (17, 18) and in mice the loss of α₂δ-3 results in aberrant synapse formation of auditory nerve fibers (19). Finally, α₂δ-4 is required for the organization of rod and cone photoreceptor synapses (20, 21).Despite these important functions, knockout mice for α₂δ-1 and α₂δ-3 show only mild neurological phenotypes (5, 10, 22–25). In contrast, mutant mice for α₂δ-2 (ducky) display impaired gait, ataxia, and epileptic seizures (26), all phenotypes consistent with a cerebellar dysfunction due to the predominant expression of α₂δ-2 in the cerebellum (e.g., ref. 15). Hence, in contrast to the specific functions of α₂δ isoforms (discussed above) the phenotypes of the available knockout or mutant mouse models suggest a partial functional redundancy in central neurons. Moreover, detailed mechanistic insights into the putative synaptic functions of α₂δ subunits are complicated by the simultaneous and strong expression of three isoforms (α₂δ-1 to -3) in neurons of the central nervous system (27).In this study, by transfecting cultured hippocampal neurons from α₂δ-2/-3 double-knockout mice with short hairpin RNA (shRNA) against α₂δ-1, we developed a cellular α₂δ subunit triple-knockout/knockdown model. Excitatory synapses from these cultures show a severe failure of synaptic vesicle recycling associated with severely reduced presynaptic calcium transients, loss of presynaptic calcium channels and presynaptic vesicle-associated proteins, and a reduced size of the presynaptic active zone (AZ). Lack of presynaptic α₂δ subunits also induces a failure of postsynaptic PSD-95 and AMPA receptor (AMPAR) localization and a thinning of the postsynaptic density (PSD). Each individual α₂δ isoform (α₂δ-1 to -3) could rescue the severe phenotype, revealing the highly redundant role of presynaptic α₂δ isoforms in glutamatergic synapse formation and differentiation. Together our results show that α₂δ subunits regulate presynaptic differentiation as well as the transsynaptic alignment of postsynaptic receptors and are thus critical for the function of glutamatergic synapses.     

Cardiac Gsα overexpression enhances L-type calcium channels through an adenylyl cyclase independent pathway

The α subunit of the stimulatory heterotrimeric G protein (G_sα) is critical for the β-adrenergic receptor activation of the cAMP messenger system. The role of G_sα in regulating cardiac Ca²⁺ channel activity, however, remains controversial. Cultured neonatal cardiac myocytes from transgenic mice overexpressing cardiac G_sα were used to assess the role of G_sα on the whole-cell Ca²⁺ currents (I_Ca). Cardiac myocytes from transgenic mice had a 490% higher peak I_Ca compared with those of either wild-type controls or G_sα-nonexpressing littermates. The effect of G_sα overexpression was mimicked by intracellular dialysis of wild-type cardiac myocytes with GTPγS-activated G_sα. This effect was not mediated by protein kinase A activation as intracellular perfusion with a protein kinase A inhibitor rendered the same degree of activation in either transgenic or wild-type myocytes also dialyzed with activated G_sα. The data indicate that G_sα overexpression is associated with a constitutive enhancement of I_Ca which is independent of the cAMP pathway and activation of endogenous adenylyl cyclase.