Distinguishing surface effects of calcium ion from pore-occupancy effects in Na+ channels

The effects of calcium ion on the Na+ activation gate were studied in squid giant axons. Saxitoxin (STX) was used to block ion entry into Na+ channels without hindering access to the membrane surface, making it possible to distinguish surface effects of calcium from pore-occupancy effects. In the presence of STX, gating kinetics were measured from gating current (Ig). The kinetic effects of external calcium concentration changes were small when STX was present. In the absence of STX, lowering the calcium concentration (from 100 to 10 mM) slowed the closing of Na+ channels (measured from INa tails) by more than a factor of 2. Surprisingly, the voltage sensitivity of closing kinetics changed with calcium concentration, and it was modified by STX. Voltage sensitivity apparently depends in part on the ability of calcium to enter and block the channels as voltage is driven negative. In external medium with no added calcium, INa tail current initially increases in amplitude severalfold with the relief of calcium block, then progressively slows and gets smaller, as calcium diffuses out of the layers investing the axon. INa tails seen just before the current disappears suggest that closing in the absence of channel block is very slow or does not occur. INa amplitude and kinetics are completely restored when calcium is returned. The results strongly suggest that calcium occupancy is a requirement for channel closing and that nonoccupied channels fold reversibly into a nonfunctional conformation.

Estrogen-induced transcription of the progesterone receptor gene does not parallel estrogen receptor occupancy

The activation of the silent endogenous progesterone receptor (PR) gene by 17-β-estradiol (E2) in cells stably transfected with estrogen receptor (ER) was used as a model system to study the mechanism of E2-induced transcription. The time course of E2-induced PR transcription rate was determined by nuclear run-on assays. No marked effect on specific PR gene transcription rates was detected at 0 and 1 h of E2 treatment. After 3 h of E2 treatment, the PR mRNA synthesis rate increased 2.0- ± 0.2-fold and continued to increase to 3.5- ± 0.4-fold by 24 h as compared with 0 h. The transcription rate increase was followed by PR mRNA accumulation. No PR mRNA was detectable at 0, 1, and 3 h of E2 treatment. PR mRNA accumulation was detected at 6 h of E2 treatment and continued to accumulate until 18 h, the longest time point examined. Interestingly, this slow and gradual transcription rate increase of the endogenous PR gene did not parallel binding of E2 to ER, which was maximized within 30 min. Furthermore, the E2–ER level was down-regulated to 15% at 3 h as compared with 30 min of E2 treatment and remained low at 24 h of E2 exposure. These paradoxical observations indicate that E2-induced transcription activation is more complicated than just an association of the occupied ER with the transcription machinery.

Molecular dynamics of a grid-mounted molecular dipolar rotor in a rotating electric field

Classical molecular dynamics is applied to the rotation of a dipolar molecular rotor mounted on a square grid and driven by rotating electric field E(ν) at T ≃ 150 K. The rotor is a complex of Re with two substituted o-phenanthrolines, one positively and one negatively charged, attached to an axial position of Rh\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{4+}}}\end{equation*}\end{document} in a [2]staffanedicarboxylate grid through 2-(3-cyanobicyclo[1.1.1]pent-1-yl)malonic dialdehyde. Four regimes are characterized by a, the average lag per turn: (i) synchronous (a < 1/e) at E(ν) = |E(ν)| > Ec(ν) [Ec(ν) is the critical field strength], (ii) asynchronous (1/e < a < 1) at Ec(ν) > E(ν) > Ebo(ν) > kT/μ, [Ebo(ν) is the break-off field strength], (iii) random driven (a ≃ 1) at Ebo(ν) > E(ν) > kT/μ, and (iv) random thermal (a ≃ 1) at kT/μ > E(ν). A fifth regime, (v) strongly hindered, W > kT, Eμ, (W is the rotational barrier), has not been examined. We find Ebo(ν)/kVcm−1 ≃ (kT/μ)/kVcm−1 + 0.13(ν/GHz)1.9 and Ec(ν)/kVcm−1 ≃ (2.3kT/μ)/kVcm−1 + 0.87(ν/GHz)1.6. For ν > 40 GHz, the rotor behaves as a macroscopic body with a friction constant proportional to frequency, η/eVps ≃ 1.14 ν/THz, and for ν < 20 GHz, it exhibits a uniquely molecular behavior.

Relationship between psychostimulant-induced "high" and dopamine transporter occupancy.

The ability of cocaine to inhibit the dopamine transporter (DAT) appears to be crucial for its reinforcing properties. The potential use of drugs that produce long-lasting inhibition of the DAT as a mean of preventing the "high" and reducing drug-seeking behavior has become a major strategy in medication development. However, neither the relation between the high and DAT inhibition nor the ability to block the high by prior DAT blockade have ever been demonstrated. To evaluate if DAT could prevent the high induced by methylphenidate (MP), a drug which like cocaine inhibits the DAT, we compared the responses in eight non-drug-abusing subjects between the first and the second of two MP doses (0.375 mg/kg, i.v.) given 60 min apart. At 60 min the high from MP has returned to baseline, but 75-80% of the drug remains in brain. Positron-emission tomography and [11C]d-threo-MP were used to estimate DAT occupancies at different times after MP. DAT inhibition by MP did not block or attenuate the high from a second dose of MP given 60 min later, despite a 80% residual transporter occupancy from the first dose. Furthermore some subjects did not perceive a high after single or repeated administration despite significant DAT blockade. These results indicate that DAT occupancy is not sufficient to account for the high, and that for DAT inhibitors to be therapeutically effective, occupancies > 80% may be required.

Modulation of promoter occupancy by cooperative DNA binding and activation-domain function is a major determinant of transcriptional regulation by activators in vivo.

Binding of transcriptional activators to a promoter is a prerequisite process in transcriptional activation. It is well established that the efficiency of activator binding to a promoter is determined by the affinity of direct interactions between the DNA-binding domain of an activator and its specific target sequences. However, I describe here that activator binding to a promoter is augmented in vivo by the effects of two other determinants that have not been generally appreciated: (i) the number of activator binding sites present in a promoter and (ii) the potency of activation domains of activators. Multiple sites within a promoter can cooperatively recruit cognate factors regardless of whether they contain an effective activation domain. This cooperativity can result in the synergistic activation of transcription. The second effect is the enhancement of activator binding to a promoter by the presence of activation domains. In this case, activation domains are not simply tethered to the promoter by the DNA-binding domain but instead assist the DNA-binding domain being tethered onto the promoter. This effect of activation domains on DNA binding is instrumental in determining how potent activators can induce steep transcriptional increases at low concentrations.

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.