Jeremy S. Treger

Photosensitivity of Neurons Enabled by Cell-Targeted Gold Nanoparticles

Unmodified neurons can be directly stimulated with light to produce action potentials, but such techniques have lacked localization of the delivered light energy. Here we show that gold nanoparticles can be conjugated to high-avidity ligands for a variety of cellular targets. Once bound to a neuron, these particles transduce millisecond pulses of light into heat, which changes membrane capacitance, depolarizing the cell and eliciting action potentials. Compared to non-functionalized nanoparticles, ligand-conjugated nanoparticles highly resist convective washout and enable photothermal stimulation with lower delivered energy and resulting temperature increase. Ligands targeting three different membrane proteins were tested; all showed similar activity and washout resistance. This suggests that many types of ligands can be bound to nanoparticles, preserving ligand and nanoparticle function, and that many different cell phenotypes can be targeted by appropriate choice of ligand. The findings have applications as an alternative to optogenetics and potentially for therapies involving neuronal photostimulation.

Resting state of the human proton channel dimer in a lipid bilayer

Significance The human proton channel hHv1 is responsible for the extrusion protons during a series of important physiological events with clinical relevance. Yet, the mechanistic details underlying voltage-dependent gating, ion selectivity, and cooperativity between homodimers remain to be determined. We carried out a structural characterization of hHv1 in a lipid bilayer using spectroscopic methods and revealed a strongly focused electric field across its putative selectivity filter. In combination with computational methods, we showed that the recently determined crystal structure of CiVSD-WT is the best structural template for dimeric hHv1, and constructed a full-length model of dimeric hHv1 with spectroscopic constraints. We proposed an explicit mechanism for hHv1 gating with functional predictions according to the one-click model recently established in CiVSD. The voltage-gated proton channel Hv1 plays a critical role in the fast proton translocation that underlies a wide range of physiological functions, including the phagocytic respiratory burst, sperm motility, apoptosis, and metastatic cancer. Both voltage activation and proton conduction are carried out by a voltage-sensing domain (VSD) with strong similarity to canonical VSDs in voltage-dependent cation channels and enzymes. We set out to determine the structural properties of membrane-reconstituted human proton channel (hHv1) in its resting conformation using electron paramagnetic resonance spectroscopy together with biochemical and computational methods. We evaluated existing structural templates and generated a spectroscopically constrained model of the hHv1 dimer based on the Ci-VSD structure at resting state. Mapped accessibility data revealed deep water penetration through hHv1, suggesting a highly focused electric field, comprising two turns of helix along the fourth transmembrane segment. This region likely contains the H+ selectivity filter and the conduction pore. Our 3D model offers plausible explanations for existing electrophysiological and biochemical data, offering an explicit mechanism for voltage activation based on a one-click sliding helix conformational rearrangement.

Real-Time Imaging of Electrical Signals with an Infrared FDA-Approved Dye

Clinical methods used to assess the electrical activity of excitable cells are often limited by their poor spatial resolution or their invasiveness. One promising solution to this problem is to optically measure membrane potential using a voltage-sensitive dye, but thus far, none of these dyes have been available for human use. Here we report that indocyanine green (ICG), an infrared fluorescent dye with FDA approval as an intravenously administered contrast agent, is voltage-sensitive. The fluorescence of ICG can follow action potentials in artificial neurons and cultured rat neurons and cardiomyocytes. ICG also visualized electrical activity induced in living explants of rat brain. In humans, ICG labels excitable cells and is routinely visualized transdermally with high spatial resolution. As an infrared voltage-sensitive dye with a low toxicity profile that can be readily imaged in deep tissues, ICG may have significant utility for clinical and basic research applications previously intractable for potentiometric dyes.

Voltage-Clamped Supported Bilayer System to Record Ion Channel Activity

Structure-function studies of voltage-dependent ion channels require controlled electrical stimuli to drive channel activity. Under voltage control, site-directed fluorescence spectroscopy is often used to probe structural rearrangements that underlie channel function. Many voltage-dependent ion channels, such as the KvAP K+ channel, must be purified from bacteria and reconstituted in a lipid membrane. The ensuing (conventional) experiment in a black lipid membrane has several problems: 1) too few channels can be measured to observe gating currents; 2) the bilayer stability is generally poor and inversely related to protein density; 3) fluorescence analysis is difficult due to channel diffusion. To circumvent these problems, we previously developed a system to voltage clamp a supported bilayer with simultaneous fluorescence imaging (Hyde et al, 2010 BPS Meeting). This system employed a 5 mm diameter supported bilayer grown atop a transparent electrode-coated coverslip. We could measure gating currents of small charged molecules and voltage-dependent responses from fluorescent membrane probes; however, we have since found that direct contact with the supporting electrode significantly hinders recordings of functional channels. We have thus introduced a self-assembled monolayer cushion grown atop a gold-coated coverslip as the support electrode. Importantly, the gold-coated coverslip is also designed to enable simultaneous fluorescence imaging of ion channels in the supported bilayer using surface plasmon-assisted microscopy. Preliminary results indicate that in response to an applied transmembrane potential, we can observe voltage-dependent fluorescence changes from S4-labeled KvAP channels consistent with presumptive gating behavior. Support: NIH GM030376; Medical Scientist National Research Service Award 5 T32 GM007281.