THE GEOTROPIC RESPONSE IN ASTERINA

Upon a surface inclined at angle α Asterina gibbosa orients upward during negatively geotropic creeping until the average angle (θ) of the path is such that Δ sin θ/Δ sin α = const. This is true also in positively geotropic movement. The direction of orientation may be temporarily reversed by mechanical disturbance. The variation of θ is greater at low slopes. Tests with directed impressed pulls, due to an attached cork float, show that the pull upon the tube feet is of primary consequence for the determination of θ. When the component of gravitational pull in the direction of movement reaches a fraction of the total pull which is proportional to the gravitational vector parallel to the surface, the laterally acting component is ineffective. On this basis, it follows that Δ sin θ/Δ sin α = const.     

Variations in the Electrostatic Landscape of Class II Human Leukocyte Antigen Molecule Induced by Modifications in the Myelin Basic Protein Peptide: A Theoretical Approach

The receptor-ligand interactions involved in the formation of the complex between Class II Major Histocompatibility Complex molecules and antigenic peptides, which are essential for establishing an adaptive immunological response, were analyzed in the Class II Human Leukocyte Antigen (HLA) - Myelin Basic Protein (MBP) peptide complex (HLA-DRβ1*1501-MBP) using a multipolar molecular electrostatic potential approach. The Human Leukocyte Antigen - peptide complex system was divided into four pockets together with their respective peptide fragment and the corresponding occupying amino acid was replaced by each of the remaining 19 amino acids. Partial atomic charges were calculated by a quantum chemistry approach at the Hatree Fock/3-21*G level, to study the behavior of monopole, dipole and quadrupole electrostatic multipolar moments. Two types of electrostatic behavior were distinguished in the pockets'' amino acids: “anchoring” located in Pocket 1 and 4, and “recognition” located in Pocket 4 and 7. According to variations in the electrostatic landscape, pockets were ordered as: Pocket 1>Pocket 9≫Pocket 4≈Pocket 7 which is in agreement with the binding ability reported for Class II Major Histocompatibility Complex pockets. In the same way, amino acids occupying the polymorphic positions β13R, β26F, β28D, β9W, β74A, β47F and β57D were shown to be key for this Receptor-Ligand interaction. The results show that the multipolar molecular electrostatic potential approach is appropriate for characterizing receptor-ligand interactions in the MHC–antigenic peptide complex, which could have potential implications for synthetic vaccine design.     

State-dependent Accessibility and Electrostatic Potential in the Channel of the Acetylcholine Receptor : Inferences from Rates of Reaction of Thiosulfonates with Substituted Cysteines in the M2 Segment of the α Subunit

Ion channel function depends on the chemical and physical properties and spatial arrangement of the residues that line the channel lumen and on the electrostatic potential within the lumen. We have used small, sulfhydryl-specific thiosulfonate reagents, both positively charged and neutral, to probe the environment within the acetylcholine (ACh) receptor channel. Rate constants were determined for their reactions with cysteines substituted for nine exposed residues in the second membrane-spanning segment (M2) of the α subunit. The largest rate constants, both in the presence and absence of ACh, were for the reactions with the cysteine substituted for αThr244, near the intracellular end of the channel. In the open state of the channel, but not in the closed state, the rate constants for the reactions of the charged reagents with several substituted cysteines depended on the transmembrane electrostatic potential, and the electrical distance of these cysteines increased from the extracellular to the intracellular end of M2. Even at zero transmembrane potential, the ratios of the rate constants for the reactions of three positively charged reagents with αT244C, αL251C, and αL258C to the rate constant for the reaction of an uncharged reagent were much greater in the open than in the closed state. This dependence of the rate constants on reagent charge is consistent with an intrinsic electrostatic potential in the channel that is considerably more negative in the open state than in the closed state. The effects of ACh on the rate constants for the reactions of substituted Cys along the length of αM2, on the dependence of the rate constants on the transmembrane potential, and on the intrinsic potential support a location of a gate more intracellular than αThr244.     

Electrostatic resistance to alpha-neurotoxins conferred by charge reversal mutations in nicotinic acetylcholine receptors

The evolution of venom resistance through coevolutionary chemical arms races has arisen multiple times throughout animalia. Prior documentation of resistance to snake venom α-neurotoxins consists of the N-glycosylation motif or the hypothesized introduction of arginine at positions 187 at the α-1 nicotinic acetylcholine receptor orthosteric site. However, no further studies have investigated the possibility of other potential forms of resistance. Using a biolayer interferometry assay, we first confirm that the previously hypothesized resistance conferred by arginine at position 187 in the honey badger does reduce binding to α-neurotoxins, which has never been functionally tested. We further discovered a novel form of α-neurotoxin resistance conferred by charge reversal mutations, whereby a negatively charged amino acid is replaced by the positively charged amino acid lysine. As venom α-neurotoxins have evolved strong positive charges on their surface to facilitate binding to the negatively charged α-1 orthosteric site, these mutations result in a positive charge/positive charge interaction electrostatically repelling the α-neurotoxins. Such a novel mechanism for resistance has gone completely undiscovered, yet this form of resistance has convergently evolved at least 10 times within snakes. These coevolutionary innovations seem to have arisen through convergent phenotypes to ultimately evolve a similar biophysical mechanism of resistance across snakes.     

Salinity Effects on Water Potential Components and Bulk Elastic Modulus of Alternanthera philoxeroides (Mart.) Griseb

Pressure volume curves for Alternanthera philoxeroides (Mart.) Griseb. (alligator weed) grown in 0 to 400 millimolar NaCl were used to determine water potential (Ψ), osmotic potential (ψ_s), turgor potential (ψ_p) and the bulk elastic modulus (ε) of shoots at different tissue water contents. Values of ψ_s decreased with increasing salinity and tissue Ψ was always lower than rhizosphere Ψ. The relationship between ψ_p and tissue water content changed because ε increased with salinity. As a result, salt-stressed plants had larger ranges of positive turgor but smaller ranges of tissue water content over which ψ_p was positive. To our knowledge, this is the first report of such a salinity effect on ε in higher plants. These increases in ε with salinity provided a mechanism by which a large difference between plant Ψ and rhizosphere Ψ, the driving force for water uptake, could be produced with relatively little water loss by the plant. A time-course study of response after salinization to 400 millimolar NaCl showed Ψ was constant within 1 day, ψ_s and ψ_p continued to change for 2 to 4 days, and ε continued to change for 4 to 12 days. Changes in ε modified the capacity of alligator weed to maintain a positive water balance and consideration of such changes in other species of higher plants should improve our understanding of salt stress.     

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.     

Interactions and Diffusion in Fine-Stranded β-lactoglobulin Gels Determined via FRAP and Binding

The effects of electrostatic interactions and obstruction by the microstructure on probe diffusion were determined in positively charged hydrogels. Probe diffusion in fine-stranded gels and solutions of β-lactoglobulin at pH 3.5 was determined using fluorescence recovery after photobleaching (FRAP) and binding, which is widely used in biophysics. The microstructures of the β-lactoglobulin gels were characterized using transmission electron microscopy. The effects of probe size and charge (negatively charged Na₂-fluorescein (376Da) and weakly anionic 70kDa FITC-dextran), probe concentration (50 to 200 ppm), and β-lactoglobulin concentration (9% to 12% w/w) on the diffusion properties and the electrostatic interaction between the negatively charged probes and the positively charged gels or solutions were evaluated. The results show that the diffusion of negatively charged Na₂-fluorescein is strongly influenced by electrostatic interactions in the positively charged β-lactoglobulin systems. A linear relationship between the pseudo-on binding rate constant and the β-lactoglobulin concentration for three different probe concentrations was found. This validates an important assumption of existing biophysical FRAP and binding models, namely that the pseudo-on binding rate constant equals the product of the molecular binding rate constant and the concentration of the free binding sites. Indicators were established to clarify whether FRAP data should be analyzed using a binding-diffusion model or an obstruction-diffusion model.     

GEOTROPIC ORIENTATION IN ARTHROPODS : III. THE FIDDLER CRAB UCA.

On an inclined surface the fiddler crab Uca pugnax, during sidewise progression, orients upward through an angle θ on the surface. The extent of negatively geotropic orientation (θ) is a rectilinear function of sin α, where α is the inclination of the surface to the horizontal. This equation differs from that describing the geotropic orientation of various other animals. The difference is traced to the fact that from an initial position with the transverse axis of the body horizontal the crab is required to turn upward to an extent such that the vertical line from its center of gravity pierces the inclined surface within the base of support provided by the legs. This leads to the equation sec θ/tan α = const., which is obeyed within the limits of precision of the measurements. This type of control of geotropic orientation represents an extension of the "muscle tension theory," and is in no sense in conflict with this view. The assumptions underlying the analytical expression connecting θ and α are verified by the asymmetry in the orientation of male fiddlers, which is shown to be due to the presence of the enlarged chela and which disappears when the claws are removed.     

TGF-β and IL-10 Production by HIV-Specific CD8+ T Cells Is Regulated by CTLA-4 Signaling on CD4+ T Cells

Immune dysregulation in HIV-1 infection is associated with increased expression of inhibitory molecules such as CTLA-4, TGF-β, and IL-10. In this study we examined one potential mechanism for regulating TGF-β and IL-10 expression by HIV-specific suppressor CD8+ T cells. No overlap between TGF-β, IL-10, and IFN-γ cytokine production by HIV-specific CD8+ T cells was observed. TGF-β positive and IL-10 positive cells were FOXP3 negative, CD25 negative, and displayed a heterogeneous surface expression of CD127. TGF-β and IL-10 positive CD8+ T cells did not express CTLA-4. Nevertheless, CTLA-4 blockade resulted in a significant decrease in HIV-specific TGF-β positive and IL-10 positive CD8+ T cell responses, and a concomitant increase in HIV-specific IFN-γ positive CD8+ T cell responses. Depletion of CD4+ T cells abrogated the impact of CTLA-4 on HIV-specific TGF-β positive and IL-10 positive CD8+ T cells. Our study suggests that CTLA-4 Signaling on CD4+ T cells regulates the inhibitory functions of the HIV-specific suppressor CD8+ T cells.     

A novel approach for predicting the uptake and toxicity of metallic and metalloid ions

Electrostatic nature of plant plasma membrane (PM) plays significant roles in the ion uptake and toxicity. Electrical potential at the PM exterior surface (ψ₀^o) influences ion distribution at the PM exterior surface, and the depolarization of ψ₀^o negativity increases the electrical driving force for cation transport, but decreases the driving force for anion transport across the PMs. Assessing environmental risks of toxic ions has been a difficult task because the ion concentration (activity) in medium is not directly corrected to its potential effects. Medium characteristics like the content of major cations have important influences on the bioavailability and toxicity of ions in natural waters and soils. Models such as the Free Ion Activity Model (FIAM) and the Biotic Ligand Model (BLM), as usually employed, neglect the ψ₀^o and hence often lead to false conclusions about interaction mechanisms between toxic ions and major cations for biology. The neglect of ψ₀^o is not inconsistent with its importance, and possibly reflects the difficulty in the measurement of ψ₀^o. Based on the dual effects of the ψ₀^o, electrostatic models were developed to better predict the uptake and toxicity of metallic and metalloid ions. These results suggest that the electrostatic models provides a more robust mechanistic framework to assess metal(loid) ecotoxicity and predict critical metal(loid) concentrations linked to a biological effect, indicating its potential utility in risk assessment of metal(loid)s in water and terrestrial ecosystems.Key words: electrostatic models, plasma membrane, surface electric potential, ion uptake, toxicity, risk assessment     

In Intact Cone Photoreceptors,a Ca2+-dependent,Diffusible Factor Modulates the cGMP-gated Ion Channels Differently than in Rods

We investigated the modulation of cGMP-gated ion channels in single cone photoreceptors isolated from a fish retina. A new method allowed us to record currents from an intact outer segment while controlling its cytoplasmic composition by superfusion of the electropermeabilized inner segment. The sensitivity of the channels to agonists in the intact outer segment differs from that measured in membrane patches detached from the same cell. This sensitivity, measured as the ligand concentration necessary to activate half-maximal currents, K _1/2, also increases as Ca²⁺ concentration decreases. In electropermeabilized cones, K _1/2 for cGMP is 335.5 ± 64.4 μM in the presence of 20 μM Ca²⁺, and 84.3 ± 12.6 μM in its absence. For 8Br-cGMP, K _1/2 is 72.7 ± 11.3 μM in the presence of 20 μM Ca²⁺ and 15.3 ± 4.5 μM in its absence. The Ca²⁺-dependent change in agonist sensitivity is larger in extent than that measured in rods. In electropermeabilized tiger salamander rods, K _1/2 for 8Br-cGMP is 17.9 ± 3.8 μM in the presence of 20 μM Ca²⁺ and 7.2 ± 1.2 μM in its absence. The Ca²⁺-dependent modulation is reversible in intact cone outer segments, but is progressively lost in the absence of divalent cations, suggesting that it is mediated by a diffusible factor. Comparison of data in intact cells and detached membrane fragments from cones indicates that this factor is not calmodulin. At 40 μM 8Br-cGMP, the Ca²⁺-dependent change in sensitivity in cones is half-maximal at K _Ca = 286 ± 66 nM Ca²⁺. In rods, by contrast, K _Ca is ∼50 nM Ca²⁺. The difference in magnitude and Ca²⁺ dependence of channel modulation between photoreceptor types suggests that this modulation may play a more significant role in the regulation of photocurrent gain in cones than in rods.     

Maximum Likelihood Estimation of Population Parameters

One of the most important parameters in population genetics is θ = 4N(e)μ where N(e) is the effective population size and μ is the rate of mutation per gene per generation. We study two related problems, using the maximum likelihood method and the theory of coalescence. One problem is the potential improvement of accuracy in estimating the parameter θ over existing methods and the other is the estimation of parameter λ which is the ratio of two θ's. The minimum variances of estimates of the parameter θ are derived under two idealized situations. These minimum variances serve as the lower bounds of the variances of all possible estimates of θ in practice. We then show that Watterson's estimate of θ based on the number of segregating sites is asymptotically an optimal estimate of θ. However, for a finite sample of sequences, substantial improvement over Watterson's estimate is possible when θ is large. The maximum likelihood estimate of λ = θ(1)/θ(2) is obtained and the properties of the estimate are discussed.     

Phosducin regulates the expression of transducin betagamma subunits in rod photoreceptors and does not contribute to phototransduction adaptation

For over a decade, phosducin's interaction with the βγ subunits of the G protein, transducin, has been thought to contribute to light adaptation by dynamically controlling the amount of transducin heterotrimer available for activation by photoexcited rhodopsin. In this study we directly tested this hypothesis by characterizing the dark- and light-adapted response properties of phosducin knockout (Pd−/−) rods. Pd−/− rods were notably less sensitive to light than wild-type (WT) rods. The gain of transduction, as measured by the amplification constant using the Lamb-Pugh model of activation, was 32% lower in Pd−/− rods than in WT rods. This reduced amplification correlated with a 36% reduction in the level of transducin βγ-subunit expression, and thus available heterotrimer in Pd−/− rods. However, commonly studied forms of light adaptation were normal in the absence of phosducin. Thus, phosducin does not appear to contribute to adaptation mechanisms of the outer segment by dynamically controlling heterotrimer availability, but rather is necessary for maintaining normal transducin expression and therefore normal flash sensitivity in rods.     

ON THE GEOTROPIC ORIENTATION OF HELIX

The snail Helix nemoralis in negatively geotropic creeping orients upward upon an inclined surface until the angle of the path of progression (θ) is related to the tilt of the surface (α) as (Δ sin θ) (Δ sin α) = – const.; θ is very nearly a rectilinear function of log sin α. The precision of orientation (P.E._θ) declines in proportion to increasing sin α, P.E._θ/^θ in proportion to θ. These facts are comprehensible only in terms of the view that the limitation of orientation is controlled by the sensorial equivalence of impressed tensions in the anterior musculature.     

THE USE OF SHADOW-CASTING TECHNIQUE FOR MEASUREMENT OF THE WIDTH OF ELONGATED PARTICLES

A method is described for the estimation of the true width of fibrillar or rod-like structures from electron micrographs of metal-shadowed preparations. The method is based on variations in the image width as a function of the angle (β) between the long axis of the fibril and the direction of the shadow in the plane of the preparation. The image width when β = 0° practically represents the real width of the elongated particle but is often indistinguishable from the background. The fibril image width is conveniently measured at β values between 15° and 90°. The true width is obtained by plotting the image width versus sin β and extrapolating to β = 0°. Latex spheres are sprayed with the fibrils or rods to indicate the direction of shadow. Tobacco mosaic virus (TMV) was used as a model structure because of its known constant diameter of 150 A (5). The width (in the case of TMV equal to the diameter) found by the present method was 150 A ± 8 A.     

Structural Insight into Amino Group-carrier Protein-mediated Lysine Biosynthesis: CRYSTAL STRUCTURE OF THE LYSZ·LYSW COMPLEX FROM THERMUS THERMOPHILUS*

In the biosynthesis of lysine by Thermus thermophilus, the metabolite α-ketoglutarate is converted to the intermediate α-aminoadipate (AAA), which is protected by the 54-amino acid acidic protein LysW. In this study, we determined the crystal structure of LysZ from T. thermophilus (TtLysZ), an amino acid kinase that catalyzes the second step in the AAA to lysine conversion, which was in a complex with LysW at a resolution of 1.85 Å. A crystal analysis coupled with isothermal titration calorimetry of the TtLysZ mutants for TtLysW revealed tight interactions between LysZ and the globular and C-terminal extension domains of the LysW protein, which were mainly attributed to electrostatic forces. These results provided structural evidence for LysW acting as a protecting molecule for the α-amino group of AAA and also as a carrier protein to guarantee better recognition by biosynthetic enzymes for the efficient biosynthesis of lysine.     

Mapping the electrostatic profiles of cellular membranes

Anionic phospholipids can confer a net negative charge on biological membranes. This surface charge generates an electric field that serves to recruit extrinsic cationic proteins, can alter the disposition of transmembrane proteins and causes the local accumulation of soluble counterions, altering the local pH and the concentration of physiologically important ions such as calcium. Because the phospholipid compositions of the different organellar membranes vary, their surface charges are similarly expected to diverge. Yet, despite the important functional implications, remarkably little is known about the electrostatic properties of the individual organellar membranes. We therefore designed and implemented approaches to estimate the surface charges of the cytosolic membranes of various organelles in situ in intact cells. Our data indicate that the inner leaflet of the plasma membrane is most negative, with a surface potential of approximately –35 mV, followed by the Golgi complex > lysosomes > mitochondria ≈ peroxisomes > endoplasmic reticulum, in decreasing order.

Lipids and (glyco)proteins are the main constituents of biological membranes. Sugar moieties of glycoproteins, glycolipids, and adherent glycocalyx components such as hyaluronic acid can bear ionizable groups that confer a net negative charge on the outer surface of the plasma membrane. The aggregate surface charge of the outer membrane has been estimated indirectly by measuring the ζ potential—the potential at the slipping plane—by electrophoretic means (e.g., Tippe, 1981; Silva Filho et al., 1987) or by measuring streaming potentials (Vandrangi et al., 2012). The plasma membrane, however, is highly asymmetric; its inner (cytosolic) aspect is virtually devoid of carbohydrate moieties. Nevertheless, the cytosolic leaflet is also thought to be negatively charged, due primarily to the accumulation of anionic phospholipids, namely phosphoinositides and phosphatidylserine (PtdSer). Based on biochemical determinations of its lipid composition, the net negative charge of the plasmalemmal inner leaflet is estimated to generate an electrical field of 10⁵ V/cm (Olivotto et al., 1996). The membranes of intracellular organelles can also contain anionic lipids, but their precise lipid composition and topology have been difficult to assess and hence their surface charge has not been estimated.The surface potentials of biological membranes have important functional implications: they can alter the disposition of charged regions of transmembrane proteins, cause local accumulation of soluble counterions in the vicinity—altering the local pH as well as the concentration of physiologically important ions such as calcium—and serve to recruit extrinsic cationic proteins (McLaughlin, 1989). It is therefore important to determine the electrostatic properties of each of the organellar membranes. In principle, this could be accomplished by measuring the ζ potentials of isolated organelles. However, the purity of such preparations is imperfect, changes in lipid composition (particularly phosphoinositide degradation) and sidedness cannot be avoided, and loosely adherent components that may alter the surface charge can be removed during the isolation process. Alternative approaches to estimating the surface potential are therefore required.Here we used recombinant and synthetic polycationic peptides to obtain a quantitative estimate of the surface potential of the inner leaflet of the plasma membrane and to establish a hierarchical map of the potentials of the cytosolic surfaces of the major intracellular organelles in live cells.     

Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex

Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14–19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.     

Mechanism for Targeting the A-kinase Anchoring Protein AKAP18δ to the Membrane

A-kinase anchoring proteins (AKAPs) are a family of scaffolding proteins that target PKA and other signaling molecules to cellular compartments and thereby spatiotemporally define cellular signaling events. The AKAP18 family comprises AKAP18α, AKAP18β, AKAP18γ, and AKAP18δ. The δ isoform targets PKA and phosphodiesterase PDE4D to AQP2 (aquaporin-2)-bearing vesicles to orchestrate the acute regulation of body water balance. Therefore, AKAP18δ must adopt a membrane localization that seems at odds with (i) its lack of palmitoylation or myristoylation sites that tailor its isoforms AKAP18α and AKAP18β to membrane compartments and (ii) the high sequence identity to the preferentially cytoplasmic AKAP18γ. Here, we show that the electrostatic attraction of the positively charged amino acids of AKAP18δ to negatively charged lipids explains its membrane targeting. As revealed by fluorescence correlation spectroscopy, the binding constant of purified AKAP18δ fragments to large unilamellar vesicles correlates (i) with the fraction of net negatively charged lipids in the bilayer and (ii) with the total amount of basic residues in the protein. Although distantly located on the sequence, these positively charged residues concentrate in the tertiary structure and form a clear binding surface. Thus, specific recruitment of the AKAP18δ-based signaling module to membranes such as those of AQP2-bearing vesicles must be achieved by additional mechanisms, most likely compartment-specific protein-protein interactions.     

Inactivation of Gating Currents of L-Type Calcium Channels : Specific Role of the α2δ Subunit

In studies of gating currents of rabbit cardiac Ca channels expressed as α_1C/β_2a or α_1C/β_2a/α₂δ subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489–517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863–895). Charge 1 (voltage of half-maximal transfer, V_1/2 ≃ 0 mV) gates noninactivated channels, while charge 2 (V_1/2 ≃ −90 mV) is generated in inactivated channels. In α_1C/β_2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant τ ≃ 8, while the available charge 2 decreased upon recovery from inactivation (at −200 mV) with τ ≃ 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of measurable charge movement and that of changes in their availability, which was even wider in the presence of α₂δ, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the α₂δ subunit did not change the V_1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to τ ≃ 2 s), while slowing the reduction of charge 2 upon recovery (τ ≃ 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489–498) and inactivation as discrete modal change. The effects of α₂δ are reproduced assuming that the subunit lowers the free energy of the inactivated mode.