Modal Gating of Human CaV2.1 (P/Q-type) Calcium Channels: I. The Slow and the Fast Gating Modes and their Modulation by β Subunits

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels and their modulation by the auxiliary β1b, β2e, β3a, and β4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human CaV2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463–474). Here, we report the characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). CaV2.1 channels containing any of the four β subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, CaV2.1 channels display different rates of inactivation and different steady-state inactivation depending on the β subtype. The type of β subunit also modulates the relative occurrence of the slow and the fast gating mode of CaV2.1 channels; β3a promotes the fast mode, whereas β4a promotes the slow mode. The prevailing mode of gating of CaV2.1 channels lacking a β subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode

Stabilization of the Cysteine-Rich Conotoxin MrIA by Using a 1,2,3-Triazole as a Disulfide Bond Mimetic

The design of disulfide bond mimetics is an important strategy for optimising cysteine-rich peptides in drug development. Mimetics of the drug lead conotoxin MrIA, in which one disulfide bond is selectively replaced of by a 1,4-disubstituted-1,2,3-triazole bridge, are described. Sequential copper-catalyzed azide–alkyne cycloaddition (CuAAC; click reaction) followed by disulfide formation resulted in the regioselective syntheses of triazole–disulfide hybrid MrIA analogues. Mimetics with a triazole replacing the Cys4–Cys13 disulfide bond retained tertiary structure and full in vitro and in vivo activity as norepinephrine reuptake inhibitors. Importantly, these mimetics are resistant to reduction in the presence of glutathione, thus resulting in improved plasma stability and increased suitability for drug development.NHMRC 1045964 & 107211

On the Universality of CP Violation in Delta F = 1 Processes

We show that new physics which breaks the left-handed SU(3)_Q quark flavor symmetry induces contributions to CP violation in Delta F = 1 couplings which are approximately universal, in that they are not affected by flavor rotations between the up and the down mass bases. (Only the short distance contributions are universal, while observables are also affected by hadronic matrix elements.) Therefore, such flavor violation cannot be aligned, and is constrained by the strongest bound from either the up or the down sectors. We use this result to show that the bound from eps'/eps prohibits an SU(3)_Q breaking explanation of the recent LHCb evidence for CP violation in D meson decays. Another consequence of this universality is that supersymmetric alignment models with a moderate mediation scale are consistent with the data, and are harder to probe via CP violating observables. With current constraints, therefore, squarks need not be degenerate. However, future improvements in the measurement of CP violation in D-Dbar mixing will start to probe alignment models.Comment: 10 pages, 2 figures. Clarifications and references adde

ERK and mTORC1 Inhibitors Enhance the Anti-Cancer Capacity of the Octpep-1 Venom-Derived Peptide in Melanoma BRAF(V600E) Mutations

Melanoma is the main cause of skin cancer deaths, with special emphasis in those cases carrying BRAF mutations that trigger the mitogen-activated protein kinases (MAPK) signaling and unrestrained cell proliferation in the absence of mitogens. Current therapies targeting MAPK are hindered by drug resistance and relapse that rely on metabolic rewiring and Akt activation. To identify new drug candidates against melanoma, we investigated the molecular mechanism of action of the Octopus Kaurna-derived peptide, Octpep-1, in human BRAF(V600E) melanoma cells using proteomics and RNAseq coupled with metabolic analysis. Fluorescence microscopy verified that Octpep-1 tagged with fluorescein enters MM96L and NFF cells and distributes preferentially in the perinuclear area of MM96L cells. Proteomics and RNAseq revealed that Octpep-1 targets PI3K/AKT/mTOR signaling in MM96L cells. In addition, Octpep-1 combined with rapamycin (mTORC1 inhibitor) or LY3214996 (ERK1/2 inhibitor) augmented the cytotoxicity against BRAF(V600E) melanoma cells in comparison with the inhibitors or Octpep-1 alone. Octpep-1-treated MM96L cells displayed reduced glycolysis and mitochondrial respiration when combined with LY3214996. Altogether these data support Octpep-1 as an optimal candidate in combination therapies for melanoma BRAF(V600E) mutations

Calculations of collisions between cold alkaline earth atoms in a weak laser field

We calculate the light-induced collisional loss of laser-cooled and trapped magnesium atoms for detunings up to 50 atomic linewidths to the red of the ^1S_0-^1P_1 cooling transition. We evaluate loss rate coefficients due to both radiative and nonradiative state-changing mechanisms for temperatures at and below the Doppler cooling temperature. We solve the Schrodinger equation with a complex potential to represent spontaneous decay, but also give analytic models for various limits. Vibrational structure due to molecular photoassociation is present in the trap loss spectrum. Relatively broad structure due to absorption to the Mg_2 ^1Sigma_u state occurs for detunings larger than about 10 atomic linewidths. Much sharper structure, especially evident at low temperature, occurs even at smaller detunings due to of Mg_2 ^1Pi_g absorption, which is weakly allowed due to relativistic retardation corrections to the forbidden dipole transition strength. We also perform model studies for the other alkaline earth species Ca, Sr, and Ba and for Yb, and find similar qualitative behavior as for Mg.Comment: 20 pages, RevTex, 13 eps figures embedde

Identifying key amino acid residues that affect alpha-conotoxin AuIB inhibition of alpha3beta4 nicotinic acetylcholine receptors

Background: -Conotoxin AuIB interacts with 34 nAChRs and GABA(B) receptors, but structural determinants of these interactions are unknown. Results: Using alanine scanning mutagenesis and molecular dynamics, we identified residues crucial for AuIB34 nAChR interaction. Conclusion: We identified the key residues that mediate AuIB34 nAChR interaction. Significance: Ability to direct -conotoxin binding to nAChRs or GABA(B) receptors will improve analgesic conopeptides

High-throughput synthesis of peptide alpha-thioesters: a safety catch linker approach enabling parallel hydrogen fluoride cleavage

Peptide -thioesters are fundamental building blocks in peptide and protein science, providing powerful tools for peptide medicinal chemistry. The application of peptide -thioesters in native chemical ligation reactions has enabled synthetic access to cysteine-rich peptides and proteins, cyclic peptides as well as labeled and chemically modified biomolecules. An efficient high-throughput synthesis of peptide -thioester building blocks would be beneficial for many medicinal chemical applications that require peptides and proteins. Herein we present a novel synthetic route to cysteine-rich peptide -thioesters using a safety catch linker that enables a parallel synthetic strategy for chemical protein synthesis. ACP(68-75), bradykinin and dynorphin(1-13) were synthesized via Boc chemistry in their thioester form on a safety catch amide linker (SCAL), employing polystyrene- or poly(ethylene glycol)-based resins, compartmentalized in tea bags. This compartmentalized resin/linker strategy facilitated a parallel hydrogen fluoride cleavage in which each peptide thioester was subsequently cyclized by native chemical ligation, demonstrating the utility of this approach. A naturally occurring bioactive cyclic peptide, the sunflower trypsin inhibitor SFTI-1, was synthesized to demonstrate the viability of this method to access important peptide biomolecules