Modulation of Mouse Rod Photoreceptor Responses by Grb14 Protein

Previous experiments have indicated that growth factor receptor-bound protein 14 (Grb14) may modulate rod photoreceptor cGMP-gated channels by decreasing channel affinity for cGMP; however, the function of Grb14 in rod physiology is not known. In this study, we examined the role of Grb14 by recording electrical responses from rods in which the gene for the Grb14 protein had been deleted. Suction-electrode recordings from single mouse rods showed that responses of dark-adapted Grb14^−/− mice to brief flashes decayed more rapidly than strain-controlled wild type (WT) rods, with decreased values of both integration time and the exponential time course of decay (τ_REC). This result is consistent with an increase in channel affinity for cGMP produced by deletion of Grb14. However, Grb14^−/− mouse rods also showed little change in dark current and a large and significant decrease in the limiting time constant τ_D, which are not consistent with an effect on channel affinity but seem rather to indicate modulation of the rate of inactivation of cyclic nucleotide phosphodiesterase 6 (PDE6). Grb14 has been reported to translocate from the inner to the outer segment in bright light, but we saw effects on response time course even in dark-adapted rods, although the effects were somewhat greater after rods had been adapted by exposure to bleaching illumination. Our results indicate that the mechanism of Grb14 action may be more complex than previously realized.     

Regulation of Mammalian Cone Phototransduction by Recoverin and Rhodopsin Kinase

Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of rhodopsin kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light.     

Calcium sets the physiological value of the dominant time constant of saturated mouse rod photoresponse recovery

Background

The rate-limiting step that determines the dominant time constant (τ_D) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca²⁺-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that τ_D is modulated by background light in mouse rods.

Methodology/Principal Findings

We used ex vivo ERG technique to record pharmacologically isolated photoreceptor responses (fast PIII component). We show a novel static effect of calcium on mouse rod phototransduction: Ca²⁺ shortens the dominant time constant (τ_D) of saturated photoresponse recovery, i.e., when extracellular free Ca²⁺ is decreased from 1 mM to ∼25 nM, the τ_D is reversibly increased ∼1.5–2-fold.

Conclusions

We conclude that the increase in τ_D during low Ca²⁺ treatment is not due to increased [cGMP], increased [Na⁺] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that a Ca²⁺-dependent mechanism controls the life time of active PDE.     

Quantitative Aspects of cGMP Phosphodiesterase Activation in Carp Rods and Cones

Cones are less light-sensitive than rods. We showed previously in carp that more light (>100-fold) is required in cones than in rods to activate 50% of cGMP phosphodiesterase (PDE). The lower effectiveness of PDE activation in carp cones is due partly to the fact that the activation rate of transducin (Tr) by light-activated visual pigment (R*) is 5-fold lower in carp cones than in rods. In this study, we tried to explain the remaining difference. First, we examined the efficiency of activation of PDE by activated Tr (Tr*). By activating PDE with known concentrations of the active (guanosine 5′-Ο-(γ-thio)triphosphate (GTPγS)-bound) form of Tr*, we found that Tr* activated PDE at a similar efficiency in rods and cones. Next, we examined the contribution of R* and Tr* lifetimes. In a comparison of PDE activation in the presence (with GTP) and absence (with GTPγS) of Tr* inactivation, PDE activation required more light (and was therefore less effective) when Tr* was inactivated in both rod and cone membranes. This is probably because inactivation of Tr* shortened its lifetime, thereby reducing the number of activated PDE molecules. The effect of Tr* inactivation was larger in cones, probably because the lifetime of Tr* is shorter in cones than in rods. The shorter lifetimes of Tr* and R* in cones seem to explain the remaining difference in the effectiveness of PDE activation between rods and cones.     

Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4

G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5′-adenylyl β,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D₁ receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.     

Functional Comparison of Rod and Cone Gαt on the Regulation of Light Sensitivity

The signaling cascades mediated by G protein-coupled receptors (GPCRs) exhibit a wide spectrum of spatial and temporal response properties to fulfill diverse physiological demands. However, the mechanisms that shape the signaling response of the GPCR are not well understood. In this study, we replaced cone transducin α (cTα) for rod transducin α (rTα) in rod photoreceptors of transgenic mice, which also express S opsin, to evaluate the role of Gα subtype on signal amplification from different GPCRs in the same cell; such analysis may explain functional differences between retinal rod and cone photoreceptors. We showed that ectopically expressed cTα 1) forms a heterotrimeric complex with rod Gβ₁γ₁, 2) substitutes equally for rTα in generating photoresponses initiated by either rhodopsin or S-cone opsin, and 3) exhibited similar light-activated translocation as endogenous rTα in rods and endogenous cTα in cones. Thus, rTα and cTα appear functionally interchangeable. Interestingly, light sensitivity appeared to correlate with the concentration of cTα when expression is reduced below 35% of normal. However, quantification of endogenous cTα concentration in cones showed a higher level to rTα in rods. Thus, reduced sensitivity in cones cannot be explained by reduced coupling efficiency between the GPCR and G protein or a lower concentration of G protein in cones versus rods.     

A Long Lasting β1 Adrenergic Receptor Stimulation of cAMP/Protein Kinase A (PKA) Signal in Cardiac Myocytes

Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β₁ adrenergic receptor (β₁AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t_½ of 3.9 h) in cardiac myocytes. However, the β₁AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β₁AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β₁AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.     

G Protein-coupled Receptor Kinase 4 (GRK4) Regulates the Phosphorylation and Function of the Dopamine D3 Receptor

During conditions of moderate sodium excess, the dopaminergic system regulates blood pressure and water and electrolyte balance by engendering natriuresis. Dopamine exerts its effects on dopamine receptors, including the dopamine D₃ receptor. G protein-coupled receptor kinase 4 (GRK4), whose gene locus (4p16.3) is linked to essential hypertension, desensitizes the D₁ receptor, another dopamine receptor. This study evaluated the role of GRK4 on D₃ receptor function in human proximal tubule cells. D₃ receptor co-segregated in lipid rafts and co-immunoprecipitated and co-localized in human proximal tubule cells and in proximal and distal tubules and glomeruli of kidneys of Wistar Kyoto rats. Bimolecular fluorescence complementation and confocal microscopy revealed that agonist activation of the receptor initiated the interaction between D₃ receptor and GRK4 at the cell membrane and promoted it intracellularly, presumably en route to endosomal trafficking. Of the four GRK4 splice variants, GRK4-γ and GRK4-α mediated a 3- and 2-fold increase in the phosphorylation of agonist-activated D₃ receptor, respectively. Inhibition of GRK activity with heparin or knockdown of GRK4 expression via RNA interference completely abolished p44/42 phosphorylation and mitogenesis induced by D₃ receptor stimulation. These data demonstrate that GRK4, specifically the GRK4-γ and GRK4-α isoforms, phosphorylates the D₃ receptor and is crucial for its signaling in human proximal tubule cells.During conditions of moderate sodium excess, the dopaminergic system sits at the fulcrum of homeostatic control of water and electrolyte balance and blood pressure (1, 2). Dopamine promotes natriuresis by inhibiting sodium chloride reabsorption in specific segments of the nephron. Dopamine exerts its action on dopamine receptors, which belong to the family of G protein-coupled receptors (GPCRs).² The dopamine receptors are classified into two subtypes based on their ability to increase cAMP levels, sequence similarity, G protein coupling, and pharmacological profiles (3, 4). The D₁-like dopamine receptors activate adenylyl cyclase by coupling to stimulatory Gα_s/Gα_olf and include the D₁ (D₁R) and D₅ receptors (D₅R). The D₂-like dopamine receptors inhibit adenylyl cyclase by coupling to Gα_i/Gα_o and consist of the D₂ (D₂R), D₃ (D₃R), and D₄ (D₄R) receptors. The D₃R has also been shown to couple to Gα_o, Gβγ, and to the stimulatory Gα_s (5, 6).The signal transduction that follows ligand occupation of a GPCR is tightly regulated to limit the specificity and extent of cellular response. GPCR-mediated signal transduction is rapidly dampened via receptor desensitization or the waning of the responsiveness of the receptor to agonist with time. Desensitization involves receptor phosphorylation and is carried out by either GPCR kinases (GRKs) or second messenger-activated kinases such as protein kinase A and protein kinase C. Homologous desensitization involves GRKs that selectively phosphorylate only agonist-activated receptors, whereas heterologous desensitization is carried out by second messenger-dependent kinases that indiscriminately phosphorylate agonist-activated receptors and those that have not been exposed to the agonist (7).The GRKs are serine/threonine protein kinases comprising seven isoforms that are grouped into three subfamilies. GRK1 and GRK7 belong to the rhodopsin kinase subfamily and are expressed exclusively in the retina (8–10). GRK2 and GRK3 phosphorylate the β-adrenergic receptor and belong to the β-adrenergic receptor kinase subfamily (11), and GRK4, GRK5, and GRK6 belong to the GRK4 subfamily. GRK4 is highly enriched in the testis and, to a lesser degree, in the kidneys (12, 13). Four splice variants of human GRK4 result from the alternative splicing of exons 2 and 15 (11). GRK4-α is considered the full-length version, whereas GRK4-β, -γ, and -δ are shortened versions of GRK4-α (14). The coding region of the GRK4 gene, whose 4p16.3 locus has been linked to essential hypertension (15, 16), contains several single nucleotide polymorphisms, including R65L, A142V, and A486V, which have been linked to essential hypertension and/or salt sensitivity in various ethnic groups (17).The D₃R gene is found at 3q13.3 (18), a locus that is also linked to essential hypertension (19, 20). Sequence analysis of the D₃R gene shows the presence of several single nucleotide polymorphisms, which do not correlate with either essential hypertension among Japanese (21) or with blood pressure levels and diabetic nephropathy among Finns (22). However, D₃R knock-out mice develop a renin-dependent form of hypertension and fail to excrete a sodium load (23).The D₃R has a long third intracellular loop that contains several putative GRK phosphorylation sites (24). A previous study evaluated the ability of GRK2 and GRK3 to phosphorylate D₃R and showed that co-transfection of GRK3, but not GRK2, resulted in a weak phosphorylation of the heterologously expressed, dopamine-stimulated D₃R in HEK-293 (25), a human embryonic kidney cell line. We tested the hypothesis that GRK4 is required in D₃R signaling in terminally differentiated human renal proximal tubule cells (hPTCs) by determining the spatiotemporal dynamics of the interaction of D₃R and GRK4 through their subfractionation in membrane microdomains and subcellular co-localization via confocal microscopy and bimolecular fluorescence complementation assay (BiFC). We also identified which of the GRK4 splice variants are involved in D₃R phosphorylation and evaluated the physiological roles of GRK4 in D₃R signaling in the hPTCs. We now report that D₃R and GRK4 co-fractionate in lipid rafts and co-localize in both hPTCs and WYK kidneys. Moreover, D₃R is phosphorylated by GRK4-γ and GRK4-α isoforms, and the absence of GRK4 impairs D₃R-mediated mitogenesis and activation of p44/42 in hPTCs.     

β-Adrenergic cAMP Signals Are Predominantly Regulated by Phosphodiesterase Type 4 in Cultured Adult Rat Aortic Smooth Muscle Cells

Background

We investigated the role of cyclic nucleotide phosphodiesterases (PDEs) in the spatiotemporal control of intracellular cAMP concentrations in rat aortic smooth muscle cells (RASMCs).

Methodology/Principal Findings

The rank order of PDE families contributing to global cAMP-PDE activity was PDE4> PDE3  =  PDE1. PDE7 mRNA expression but not activity was confirmed. The Fluorescence Resonance Energy Transfer (FRET)-based cAMP sensor, Epac1-camps, was used to monitor the time course of cytosolic cAMP changes. A pulse application of the β-adrenoceptor (β-AR) agonist isoproterenol (Iso) induced a transient FRET signal. Both β₁- and β₂-AR antagonists decreased the signal amplitude without affecting its kinetics. The non-selective PDE inhibitor (IBMX) dramatically increased the amplitude and delayed the recovery phase of Iso response, in agreement with a role of PDEs in degrading cAMP produced by Iso. Whereas PDE1, PDE3 and PDE7 blockades [with MIMX, cilostamide (Cil) and BRL 50481 (BRL), respectively] had no or minor effect on Iso response, PDE4 inhibition [with Ro-20-1724 (Ro)] strongly increased its amplitude and delayed its recovery. When Ro was applied concomitantly with MIMX or Cil (but not with BRL), the Iso response was drastically further prolonged. PDE4 inhibition similarly prolonged both β₁- and β₂-AR-mediated responses. When a membrane-targeted FRET sensor was used, PDE3 and PDE4 acted in a synergistic manner to hydrolyze the submembrane cAMP produced either at baseline or after β-AR stimulation.

Conclusion/Significance

Our study underlines the importance of cAMP-PDEs in the dynamic control of intracellular cAMP signals in RASMCs, and demonstrates the prominent role of PDE4 in limiting β-AR responses. PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, and acts synergistically with PDE3 inhibition at the submembrane compartment. This suggests that mixed PDE4/PDE1 or PDE4/PDE3 inhibitors would be attractive to potentiate cAMP-related functions in vascular cells.     

Dynamic Light-Scattering Evidence for the Flexibility of Native Muscle Thin Filaments

We have obtained clear evidence for the flexibility of native scallop adductor thin filaments by studying the temperature and ionic strength dependence of the average decay constants obtained from intensity fluctuation spectroscopic (IFS) measurements. The low-angle (10-25°), average decay constants obtained from time autocorrelation functions of scattered light were independent of concentration (0.08-1.3 mg/ml), scaled with the ratio of temperature to solvent viscosity, T/η, over a range of 4-45°C, and yielded a value for the translational diffusion coefficient of D_T^5°C = (1.24 ± 0.06) × 10^-8 cm²/s. From this value and the Broersma relation for rigid rods, we find an average filament length of 1.06 ± 0.06 μm. Quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that at high temperatures (> 35°C) or in 0.6 M NaCl, tropomyosin completely dissociates from native thin filaments. Decay constants from high-angle (60-150°C) IFS temperature dependence measurements do not scale with T/η and hence do not show the temperature dependence expected for rigid rods. The differences are not due to any change in length distribution of filaments with temperature or to the free tropomyosin in solution, but are attributed to nonrigid motions of the filaments. Similar experiments on samples in high- and low-salt solvents gave results consistent with this interpretation.     

Mapping the Putative G Protein-coupled Receptor (GPCR) Docking Site on GPCR Kinase 2: INSIGHTS FROM INTACT CELL PHOSPHORYLATION AND RECRUITMENT ASSAYS*

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α_2A-adrenergic receptors (α_2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α_2AAR with EC₅₀ values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu⁴, Val⁷, Leu⁸, Val¹¹, and Ser¹², directly interacts with receptors, whereas residues such as Asp¹⁰, Tyr¹³, Ala¹⁶, Met¹⁷, Gly⁴⁷⁵, Val⁴⁷⁷, and Ile⁴⁸⁵ are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.     

The Cell Adaptation Time Sets a Minimum Length Scale for Patterned Substrates

The structure and dynamics of tissue cultures depend strongly on the physical and chemical properties of the underlying substrate. Inspired by previous advances in the context of inorganic materials, the use of patterned culture surfaces has been proposed as an effective way to induce space-dependent properties in cell tissues. However, cells move and diffuse, and the transduction of external stimuli to biological signals is not instantaneous. Here, we show that the fidelity of patterns to demix tissue cells depends on the relation between the diffusion (τ_D) and adaptation (τ) times. Numerical results for the self-propelled Voronoi model reveal that the fidelity decreases with τ/τ_D, a result that is reproduced by a continuum reaction-diffusion model. Based on recent experimental results for single cells, we derive a minimal length scale for the patterns in the substrate that depends on τ/τ_D and can be much larger than the cell size.     

The unstructured C-terminus of the tau subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the alpha subunit

The τ subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the α subunit through its C-terminal Domain V, τ_C16. We show that the extreme C-terminal region of τ_C16 constitutes the site of interaction with α. The τ_C16 domain, but not a derivative of it with a C-terminal deletion of seven residues (τ_C16Δ7), forms an isolable complex with α. Surface plasmon resonance measurements were used to determine the dissociation constant (K_D) of the α−τ_C16 complex to be ~260pM. Competition with immobilized τ_C16 by τ_C16 derivatives for binding to α gave values of K_D of 7μM for the α−τ_C16Δ7 complex. Low-level expression of the genes encoding τ_C16 and τ_C167, but not τ_C16Δ11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3′ end of the τ_C16 gene, that led to defects in α binding. The data suggest that the unstructured C-terminus of τ becomes folded into a helix–loop–helix in its complex with α. An N-terminally extended construct, τ_C24, was found to bind DNA in a salt-sensitive manner while no binding was observed for τ_C16, suggesting that the processivity switch of the replisome functionally involves Domain IV of τ.     

A Truncated Form of Rod Photoreceptor PDE6 β-Subunit Causes Autosomal Dominant Congenital Stationary Night Blindness by Interfering with the Inhibitory Activity of the γ-Subunit

Autosomal dominant congenital stationary night blindness (adCSNB) is caused by mutations in three genes of the rod phototransduction cascade, rhodopsin (RHO), transducin α-subunit (GNAT1), and cGMP phosphodiesterase type 6 β-subunit (PDE6B). In most cases, the constitutive activation of the phototransduction cascade is a prerequisite to cause adCSNB. The unique adCSNB-associated PDE6B mutation found in the Rambusch pedigree, the substitution p.His258Asn, leads to rod photoreceptors desensitization. Here, we report a three-generation French family with adCSNB harboring a novel PDE6B mutation, the duplication, c.928-9_940dup resulting in a tyrosine to cysteine substitution at codon 314, a frameshift, and a premature termination (p.Tyr314Cysfs*50). To understand the mechanism of the PDE6β1-314fs*50 mutant, we examined the properties of its PDE6-specific portion, PDE6β1-313. We found that PDE6β1-313 maintains the ability to bind noncatalytic cGMP and the inhibitory γ-subunit (Pγ), and interferes with the inhibition of normal PDE6αβ catalytic subunits by Pγ. Moreover, both truncated forms of the PDE6β protein, PDE6β1-313 and PDE6β1-314fs*50 expressed in rods of transgenic X. laevis are targeted to the phototransduction compartment. We hypothesize that in affected family members the p.Tyr314Cysfs*50 change results in the production of the truncated protein, which binds Pγ and causes constitutive activation of the phototransduction thus leading to the absence of rod adaptation.     

Coordinated control of sensitivity by two splice variants of Gαo in retinal ON bipolar cells

The high sensitivity of scotopic vision depends on the efficient retinal processing of single photon responses generated by individual rod photoreceptors. At the first synapse in the mammalian retina, rod outputs are pooled by a rod “ON” bipolar cell, which uses a G-protein signaling cascade to enhance the fidelity of the single photon response under conditions where few rods absorb light. Here we show in mouse rod bipolar cells that both splice variants of the G_o α subunit, Gα_o1 and Gα_o2, mediate light responses under the control of mGluR6 receptors, and their coordinated action is critical for maximizing sensitivity. We found that the light response of rod bipolar cells was primarily mediated by Gα_o1, but the loss of Gα_o2 caused a reduction in the light sensitivity. This reduced sensitivity was not attributable to the reduction in the total number of G_o α subunits, or the altered balance of expression levels between the two splice variants. These results indicate that Gα_o1 and Gα_o2 both mediate a depolarizing light response in rod bipolar cells without occluding each other’s actions, suggesting they might act independently on a common effector. Thus, Gα_o2 plays a role in improving the sensitivity of rod bipolar cells through its action with Gα_o1. The coordinated action of two splice variants of a single Gα may represent a novel mechanism for the fine control of G-protein activity.     

Mathematical analysis of the dependence of cell potential on external potassium in corn roots

The K⁺ dependence of normal (ψ) and diffusion (ψ_D) potentials in corn roots [Zea mays L., hybrid (A619 × Oh43) × A632] was determined experimentally and analyzed with respect to the parameter ξ [defined as exp (F ψ/RT)]. In the presence of 10 micromolar carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), ψ behaved as expected of a diffusion potential. Based upon the assumptions (a) that FCCP did not change any term of the Goldman-Hodgkin-Katz equation, and (b) that total potential was functionally the algebraic sum of ψ_D and ψ_P (the deviation from ψ_D due to an electrogenic system), ψ_P was found to be a complex function of external potassium and to have a minimum value of 0.69 millimolar K ion activity outside the cell. Analysis of ψ allowed us to develop an equation which predicts a complicated K⁺ dependence of ψ such as that found by Mertz and Higinbotham (Membrane Transport in Plants and Plant Organelles. Springer-Verlag 1974).     

High genetic diversity at the extreme range edge: nucleotide variation at nuclear loci in Scots pine (Pinus sylvestris L.) in Scotland

Nucleotide polymorphism at 12 nuclear loci was studied in Scots pine populations across an environmental gradient in Scotland, to evaluate the impacts of demographic history and selection on genetic diversity. At eight loci, diversity patterns were compared between Scottish and continental European populations. At these loci, a similar level of diversity (θ_sil=∼0.01) was found in Scottish vs mainland European populations, contrary to expectations for recent colonization, however, less rapid decay of linkage disequilibrium was observed in the former (ρ=0.0086±0.0009, ρ=0.0245±0.0022, respectively). Scottish populations also showed a deficit of rare nucleotide variants (multi-locus Tajima''s D=0.316 vs D=−0.379) and differed significantly from mainland populations in allelic frequency and/or haplotype structure at several loci. Within Scotland, western populations showed slightly reduced nucleotide diversity (π_tot=0.0068) compared with those from the south and east (0.0079 and 0.0083, respectively) and about three times higher recombination to diversity ratio (ρ/θ=0.71 vs 0.15 and 0.18, respectively). By comparison with results from coalescent simulations, the observed allelic frequency spectrum in the western populations was compatible with a relatively recent bottleneck (0.00175 × 4N_e generations) that reduced the population to about 2% of the present size. However, heterogeneity in the allelic frequency distribution among geographical regions in Scotland suggests that subsequent admixture of populations with different demographic histories may also have played a role.     

On the Origin and the Signal-Shaping Mechanism of the Fast Photosignal in the Vertebrate Retina

Fast photosignals (FPS) with R₁ and R₂ components were measured in retinas of cattle, rat, and frog within a temperature range of 0° to 60°C. Except for temperatures near 0°C the signal rise of the R₁ component was determined by the duration of the exciting flash. The kinetics of the R₂ component and the meta transition of rhodopsin in the cattle and rat retina were compared. For the analysis of the FPS it is presupposed that the signal is produced by light-induced charges on the outer segment envelope membrane that spread onto the whole plasma membrane of the photoreceptor cell. To a good approximation, this mechanism can be described by a model circuit with two distinct capacitors. In this model, the charging capacitance of the pigmented outer segment envelope membrane and the capacitance of the receptor's nonpigmented plasma membrane are connected via the extra- and intracellular electrolyte resistances. The active charging is explained by two independent processes, both with exponential rise (R₁ and R₂), that are due to charge displacements within the pigmented envelope membrane. The time constant τ₂ of the R₂ membrane charging process shows a strong temperature dependence that of the charge redistribution, τ_r, a weak one. In frog and cattle retinas the active charging is much slower within a large temperature range than the passive charge redistribution. From the two-capacitor model it follows for τ_r « τ₂ that the rise of the R₂ component is determined by τ_r, whereas the decay is given by τ₂. For the rat retina, however, τ₂ approaches τ_r at physiological temperatures and becomes <τ_r above 45°C. In this temperature range where τ₂ ≈ τ_r, both processes affect rise and decay of the photosignal. The absolute values of τ_r are in good accordance with the known electric parameters of the photoreceptors. At least in the cattle retina, the time constant τ₂ is identical with that of the slow component of the meta II formation. The strong temperature dependence of the meta transition time gives rise to the marked decrease of the R₂ amplitude with falling temperature. As the R₁ rise could not be fully time resolved the signal analysis does not yield the time constant τ₁ of the R₁ generating process. It could be established, however, within the whole temperature range that the decay of the R₁ component is determined by τ_r. Using an extended model that allows for membrane leakage, we show that in normal ringer solution the membrane time constant does not influence the signal time-course and amplitude.     

Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D₁-like family of dopamine receptors and inputs of aversion coming from neurons containing the D₂-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D₁ reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D₂ receptors function, we present evidence of σ₁ receptor molecular and functional interaction with dopamine D₂ receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D₂ receptors (the long isoform of the D₂ receptor) can complex with σ₁ receptors, a result that is specific to D₂ receptors, as D₃ and D₄ receptors did not form heteromers. We demonstrate that the σ₁-D₂ receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ₁ -D₂ receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ₁ knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D₂ receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D₁ and D₂ receptor containing neurons in the brain.     

Mechanism of response of potential-sensitive dyes studied by time-resolved fluorescence

The mechanism of response of two potential-sensitive dyes, diOC₂(5) (3,3′-diethyloxadicarbocyanine iodide) and oxonol V (bis-[3-phenyl-5-oxoisoxazol-4-yl]pentamethine oxonol), were studied by using steady-state and time-resolved fluorescence techniques. The lipid concentration dependence of the Δψ (membrane potential)-induced change in total fluorescence intensity was quite different for these two dyes. Time-resolved fluorescence measurements showed that the fluorescence decay of these dyes in membranes could be resolved into at least three exponentials. Δψ-induced changes in the levels of these three populations were also measured under a variety of conditions. In the case of diOC₂(5) an inside negative Δψ increased the levels of the bound forms. This shows that diOC₂(5) responds to Δψ mainly by an “on-off” mechanism whereby Δψ perturbs the membrane-water partition coefficient of the dye. The Δψ-induced changes approached zero when the dye was totally membrane bound. In contrast, the Δψ-induced response of oxonol V increased with increased membrane binding. An inside negative Δψ decreased the level of the bound form with a longer lifetime. This shows that the mechanism of response of oxonol V is a Δψ-induced shift in the equilibrium between bound forms of the dye.