Clinically there is a need for local anesthetics with a greater specificity of action on target cells and longer duration. We have synthesized a series of local anesthetic derivatives we call boronicaines in which the aromatic phenyl ring of lidocaine was replaced with ortho-, meta-, C,C'-dimethyl meta- and para-carborane clusters. The boronicaine derivatives were tested for their analgesic activity and compared with lidocaine using standard procedures in mice following a plantar injection. The compounds differed in their analgesic activity in the following order: ortho-carborane = C,C'-dimethyl meta-carborane > para-carborane > lidocaine > meta-carborane derivative. Both ortho-boronicaine and C,C'-dimethyl meta-boronicaine had longer durations of analgesia than lidocaine. Differences in analgesic efficacies are rationalized by variations in chemical structure and protein binding characteristics.

As a continuation of work on metallacarborane-based molecular motors, the structures of substituted bis(dicarbollyl)nickel complexes in Ni(III) and Ni(IV) oxidation states were investigated in solution by fluorescence spectroscopy. Symmetrically positioned cage-linked pyrene molecules served as fluorescent probes to enable the observation of mixed meso-trans/dl-gauche (pyrene monomer fluorescence) and dl-cis/dl-gauche (intramolecular pyrene excimer fluorescence with residual monomer fluorescence) cage conformations of the nickelacarboranes in the Ni(III) and Ni(IV) oxidation states, respectively. The absence of energetically disfavored conformers in solution--dl-cis in the case of nickel(III) complexes and meso-trans in the case of nickel(IV)--was demonstrated based on spectroscopic data and conformer energy calculations in solution. The conformational persistence observed in solution indicates that bis(dicarbollyl)nickel complexes may provide attractive templates for building electrically driven and/or photodriven molecular motors.

Channelrhodopsins (ChR1 and ChR2) are light-activated ion channels that enable photomobility of microalgae from the genus Chlamydomonas. Despite common use of ChR2 in optogenetics for selective control and monitoring of individual neurons in living tissue, the protein structures remain unresolved. Instead, a crystal structure of the ChR chimera (C1C2), an engineered combination of helices I-V from ChR1, without its C-terminus, and helices VI-VII from ChR2, is used as a template for ChR2 structure prediction. Surprisingly few studies have focused in detail on the chimera. Here, we present atomistic molecular dynamics studies of the closed-state, non-conducting C1C2 structure and protonation states. A new and comprehensive characterization of interactions in the vicinity of the gating region of the pore, namely between residues E90, E123, D253, N258, and the protonated Schiff base (SBH), as well as nearby residues K93, T127, and C128, indicates that the equilibrated C1C2 structure with both E123 and D253 deprotonated closely resembles the available crystal structure. In agreement with experimental studies on C1C2, no direct or water-mediated hydrogen bonding between an aspartate and a cysteine (D156-O…S-C128) that would define a direct-current gate in C1C2 was observed in our simulations. Finally, we show that a single hydrogen bond between a glutamic acid (E90) and an asparagine (N258) residue suffices to keep the gate of C1C2 closed and to disable free water and ion passage through the putative pore, in contrast to the double bond proposed earlier for ChR2. We anticipate that this work will provide context for studies of both the gating process and water and ion transport in C1C2, and will spark interest in further experimental studies on the chimera.

No abstract

No abstract

Channelrhodopsins (ChR) are cation channels that can be expressed heterologously in various living tissues, including cardiac and neuronal cells. To tune spatial and temporal control of potentials across ChR-enriched cell membranes, it is essential to understand how pore hydration impacts the ChR photocycle kinetics. Here, we measure channel opening and closing rates of channelrhodopsin chimera and selected variants (C1C2 wild type, C1C2-N297D, C1C2-N297V, and C1C2-V125L) and correlate them with changes in chemical interactions among functionally important residues in both closed and open states. Kinetic results substantiate that replacement of helices I and II in ChR2 with corresponding residues from ChR1, to make the chimera C1C2, affects the kinetics of channelrhodopsin pore gating significantly, making C1C2 a unique channel. As a prerequisite for studies of ion transport, detailed understanding of the water pathway within a ChR channel is important. Our atomistic simulations confirm that opening of the channel and initial hydration of the previously dry gating regions between helices I, II, III, and VII of the channel occurs with 1) the presence of 13-cis retinal; 2) deprotonation of a glutamic acid gating residue, E129; and 3) subsequent weakening of the central gate hydrogen bond between the same glutamic acid E129 and asparagine N282 in the central region of the pore. Also, an aspartate (D292) is the unambiguous primary proton acceptor for the retinal Schiff base in the hydrated channel. SIGNIFICANCEChannelrhodopsins (ChR) are light-sensitive ion channels used in optogenetics, a technique that applies light to selectively and non-invasively control cells (e.g., neurons) that have been modified genetically to express those channels. Using electrophysiology, we measured the opening and closing rates of a ChR chimera, and several variants, and correlated those rates with changes in chemical interactions determined from atomistic simulations. Significant new insights include correlation of single-point-mutations with four factors associated with pore hydration and cation conductance. Additionally, our work unambiguously identifies the primary proton acceptor for the retinal chromophore in the channel open state. These new insights add to mechanistic understanding of lightgated membrane transport and should facilitate future efforts to control membrane potentials spatially and temporally in optogenetics.

No abstract

Understanding the mechanism of toxicity of nanoparticles (NPs) represents a major challenge for biomedical application. Since the cardiotoxicity of drugs is mainly due to blockade of the human ether-a-go-go related gene (hERG) protein, the inhibition of hERG channel is a first step in a non-clinical testing strategy. Superparamagnetic iron oxide nanoparticles (SPIONs) are composed of either a magnetite (Fe 3 O 4 ) or maghemite (g-Fe 2 O 3 ) core coated with a biocompatible polymer. Both maghemite and magnetite are ferrimagnetic in nature, and when the size of the particles enters the nanometric scale (below ca. 80-120 nm) they assume a single domain magnetic structure and superparamagnetic feature. SPIONs are suitable for biological application for the following reasons: i) Fe is a naturally occurring metal in the human body; ii) they can be utilized by the body in subsequent metabolic processes; iii) their magnetic behavior allows them to be used in various biomedical applications, e.g. magnetic fluid hyperthermia. In this work we describe the interaction of SPIONs, coated with different ligands, with hERG channel. The NPs were synthesized by thermal decomposition and investigated by XRD, TEM and DLS analysis; the superparamagnetic behavior was confirmed by SQUID magnetometric analysis. We functionalized the NPs by a coating exchange reaction with several ligands and analyzed them by DLS and Z-Potential analysis. The interaction between SPIONs and hERG channel, expressed in HEK cells, was investigated by patch-clamp. We found that NPs coated with polyacrylic acid (PAA) or 2,3-dimercaptosuccinic acid (DMSA) blocked hERG channels. On the contrary, 3-aminopropyl phosphonic acid (APPA)-stabilized NPs or non-coated NPs had no effect on hERG channel, suggesting an important role of charge density of NPs in hERG binding interactions. A mode of action fitting the experimental evidence has been proposed for the observed hERG blocking activity.