Bezanilla Lab
This lab is interested in the search for the dynamics of the molecular correlates of the function in membrane transport proteins. This is being approached with physical techniques such as temperature effects and complex capacitance measurements in the frequency domain combined with mutations of the molecule and assessed by gating currents, macroscopic currents and single molecule recordings. The correlation with structural changes are being monitored with optical techniques using real time fluorescence spectroscopy including lifetimes, changes in intensity and fluorescence resonance energy transfer from probes attached to strategic sites in the molecule of interest while being functional in the membrane.`
Francisco Bezanilla
Ben Fosque
Bernardo Pinto
Hlafira Polishchuk
Yichen Liu
Miguel Angel Fernandez
Recent
Photolipid excitation triggers depolarizing optocapacitive currents and action potentials (2023)
Optically-induced changes in membrane capacitance may regulate neuronal activity without requiring genetic modifications. Previously, they mainly relied on sudden temperature jumps due to light absorption by membrane-associated nanomaterials or water. Yet, nanomaterial targeting or the required high infrared light intensities obstruct broad applicability. Now, scholars propose a very versatile approach: photolipids (azobenzene-containing diacylglycerols) mediate light-triggered cellular de- or hyperpolarization. As planar bilayer experiments show, the respective currents emerge from millisecond-timescale changes in bilayer capacitance. UV light changes photolipid conformation, which awards embedding plasma membranes with increased capacitance and evokes depolarizing currents. They open voltage-gated sodium channels in cells, generating action potentials. Blue light reduces the area per photolipid, decreasing membrane capacitance and eliciting hyperpolarization. If present, mechanosensitive channels respond to the increased mechanical membrane tension, generating large depolarizing currents that elicit action potentials. Membrane self-insertion of administered photolipids and focused illumination allows cell excitation with high spatiotemporal control.
Nongenetic optical neuromodulation with silicon-based materials (2019)
Optically controlled nongenetic neuromodulation represents a promising approach for the fundamental study of neural circuits and the clinical treatment of neurological disorders. Among the existing material candidates that can transduce light energy into biologically relevant cues, silicon (Si) is particularly advantageous due to its highly tunable electrical and optical properties, ease of fabrication into multiple forms, ability to absorb a broad spectrum of light, and biocompatibility. This protocol describes a rational design principle for Si-based structures, general procedures for material synthesis and device fabrication, a universal method for evaluating material photoresponses, detailed illustrations of all instrumentation used, and demonstrations of optically controlled nongenetic modulation of cellular calcium dynamics, neuronal excitability, neurotransmitter release from mouse brain slices, and brain activity in the mouse brain in vivo using the aforementioned Si materials. The entire procedure takes ~4–8 d in the hands of an experienced graduate student, depending on the specific biological targets. Scholars anticipate that their approach can also be adapted in the future to study other systems, such as cardiovascular tissues and microbial communities.
Cholesterol Functionalization of Gold Nanoparticles Enhances Photoactivation of Neural Activity (2018)
Gold nanoparticles (AuNPs) attached to the extracellular plasma membrane enable action potential (AP) generation in response to light. Binding AuNP bioconjugates to membrane proteins allows robust AP generation, but a non-protein-specific approach could simplify establishing light-responsiveness in excitable neurons. This study tests whether AuNPs functionalized with cholesterol (AuNP–PEG–Chol) enable light-induced APs. Dorsal root ganglion (DRG) neurons labeled with 20 nm AuNP–PEG–Chol conjugates responded to 532 nm light pulses, likely due to plasmonic heating-induced depolarization. Similar effects were observed with intracellular delivery, demonstrating a simple, stable method for inducing photosensitivity in neurons.
Photoelectrochemical modulation of neuronal activity with free-standing coaxial silicon nanowires (2018)
Optical methods for modulating cellular behaviour are promising for both fundamental and clinical applications. However, most available methods are either mechanically invasive, require genetic manipulation of target cells or cannot provide subcellular specificity. Here, we address all these issues by showing optical neuromodulation with free-standing coaxial p-type/intrinsic/n-type silicon nanowires. We reveal the presence of atomic gold on the nanowire surfaces, likely due to gold diffusion during the material growth. To evaluate how surface gold impacts the photoelectrochemical properties of single nanowires, we used modified quartz pipettes from a patch clamp and recorded sustained cathodic photocurrents from single nanowires. We show that these currents can elicit action potentials in primary rat dorsal root ganglion neurons through a primarily atomic gold-enhanced photoelectrochemical process.