João L. Carvalho-de-Souza

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.

Heterogeneous silicon mesostructures for lipid-supported bioelectric interfaces

Silicon-based materials have widespread application as biophysical tools and biomedical devices. Here we introduce a biocompatible and degradable mesostructured form of silicon with multiscale structural and chemical heterogeneities. The material was synthesized using mesoporous silica as a template through a chemical-vapor-deposition process. It has an amorphous atomic structure, an ordered nanowire-based framework, and random submicrometre voids, and shows an average Young’s modulus that is 2–3 orders of magnitude smaller than that of single crystalline silicon. In addition, we used the heterogeneous silicon mesostructures to design a lipid-bilayer-supported bioelectric interface that is remotely controlled and temporally transient, and that permits non-genetic and subcellular optical modulation of the electrophysiology dynamics in single dorsal root ganglia neurons. Our findings suggest that the biomimetic expansion of silicon into heterogeneous and deformable forms can open up opportunities in extracellular biomaterial or bioelectric systems.

Eugenol modifies the excitability of rat sciatic nerve and superior cervical ganglion neurons

Eugenol is a phenylpropene obtained from the essential oils of plants such as clove and basil which has ample use in dentistry. Eugenol possesses analgesic effects that may be related to the inhibition of voltage-dependent Na+ channels and/or to the activation of TRPV1 receptors or both. In the present study, electrophysiological parameters were taken from the compound action potentials of the isolated rat sciatic nerve and from neurons of the superior cervical ganglion (SCG) impaled with sharp microelectrodes under current-clamp conditions. In the isolated rat sciatic nerve, eugenol inhibited the compound action potential in a concentration-dependent manner. Action potentials recorded from SCG neurons were inhibited by eugenol with an IC(50) of 0.31 mM. At high concentrations (2 mM), during brief applications, eugenol caused significant action potential blockade while it did not interfere with the resting membrane potential or the membrane input resistance. Surprisingly, however, at low eugenol concentrations (0.6 mM), during long time applications, a reversible reduction (by about 50%) in the input membrane resistance was observed, suggesting the possible involvement of a secondary delayed effect of eugenol to reduce neuronal excitability.

Optocapacitive Generation of Action Potentials by Microsecond Laser Pulses of Nanojoule Energy

Millisecond pulses of laser light delivered to gold nanoparticles residing in close proximity to the surface membrane of neurons can induce membrane depolarization and initiate an action potential. An optocapacitance mechanism proposed as the basis of this effect posits that the membrane-interfaced particle photothermally induces a cell-depolarizing capacitive current, and predicts that delivering a given laser pulse energy within a shorter period should increase the pulse’s action-potential-generating effectiveness by increasing the magnitude of this capacitive current. Experiments on dorsal root ganglion cells show that, for each of a group of interfaced gold nanoparticles and microscale carbon particles, reducing pulse duration from milliseconds to microseconds markedly decreases the minimal pulse energy required for AP generation, providing strong support for the optocapacitance mechanism hypothesis.

Photoelectrochemical modulation of neuronal activity with free-standing coaxial silicon nanowires

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.The wireless and photoelectrochemical stimulation of primary rat dorsal root ganglion neurons is demonstrated by shining laser light onto coaxially doped silicon nanowires deposited on the neuronal membrane.

Nonsensing residues in S3–S4 linker’s C terminus affect the voltage sensor set point in K+ channels

Voltage sensitivity in ion channels is a function of highly conserved arginine residues in their voltage-sensing domains (VSDs), but this conservation does not explain the diversity in voltage dependence among different K+ channels. Here we study the non–voltage-sensing residues 353 to 361 in Shaker K+ channels and find that residues 358 and 361 strongly modulate the voltage dependence of the channel. We mutate these two residues into all possible remaining amino acids (AAs) and obtain Q-V and G-V curves. We introduced the nonconducting W434F mutation to record sensing currents in all mutants except L361R, which requires K+ depletion because it is affected by W434F. By fitting Q-Vs with a sequential three-state model for two voltage dependence–related parameters (V0, the voltage-dependent transition from the resting to intermediate state and V1, from the latter to the active state) and G-Vs with a two-state model for the voltage dependence of the pore domain parameter (V1/2), Spearman’s coefficients denoting variable relationships with hydrophobicity, available area, length, width, and volume of the AAs in 358 and 361 positions could be calculated. We find that mutations in residue 358 shift Q-Vs and G-Vs along the voltage axis by affecting V0, V1, and V1/2 according to the hydrophobicity of the AA. Mutations in residue 361 also shift both curves, but V0 is affected by the hydrophobicity of the AA in position 361, whereas V1 and V1/2 are affected by size-related AA indices. Small-to-tiny AAs have opposite effects on V1 and V1/2 in position 358 compared with 361. We hypothesize possible coordination points in the protein that residues 358 and 361 would temporarily and differently interact with in an intermediate state of VSD activation. Our data contribute to the accumulating knowledge of voltage-dependent ion channel activation by adding functional information about the effects of so-called non–voltage-sensing residues on VSD dynamics.

Nav channel binder containing a specific conjugation-site based on a low toxicity β-scorpion toxin

Voltage-gated sodium (Nav) channels play a key role in generating action potentials which leads to physiological signaling in excitable cells. The availability of probes for functional studies of mammalian Nav is limited. Here, by introducing two amino acid substitutions into the beta scorpion toxin Ts1, we have chemically synthesized a novel binder [S14R, W50Pra]Ts1 for Nav with high affinity, low dissociation rate and reduced toxicity while retaining the capability of conjugating Ts1 with molecules of interests for different applications. Using the fluorescent-dye conjugate, [S14R, W50Pra(Bodipy)]Ts1, we confirmed its binding to Nav1.4 through Lanthanide-based Resonance Energy Transfer. Moreover, using the gold nanoparticle conjugate, [S14R, W50Pra(AuNP)]Ts1, we were able to optically stimulate dorsal root ganglia neurons and generate action potentials with visible light via the optocapacitive effect as previously reported. [S14R, W50Pra]Ts1 is a novel probe with great potential for wider applications in Nav-related neuroscience research.

Roadmap on semiconductor–cell biointerfaces

This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world.

Insights on the Mechanisms of the Fast Blockade of TTX-R Na+ Channels by Eugenol

OBJECTIVES. It was previously shown that eugenol, a phenylpropene, blocks fast and reversibly voltage-gated Na+ channels (NaV), but little concern was given to the blocker binding to different conformational states of channel molecule. Here we reported a detailed analysis of state-dependent effects of eugenol on tetrodotoxin-resistant (TTX-R) NaV isoforms, comparing them to those of lidocaine, a reference blocker.METHODS. TTX-R Na+ currents were recorded in dorsal root ganglia neurons from newborn Wistar rats with whole-cell configuration of patch clamp technique. Tetrodotoxin-sensitive Na+ currents were blocked by TTX 100nM in the extracellular solution.RESULTS and CONCLUSIONS. A dose-dependent fast blockade due to eugenol was observed in 0.2Hz time series depolarizations from a holding potential of −110 mV to a 0 mV pulse. This tonic blockage is due to eugenol binding to the closed state. The IC50 was 2.28±0.10mM for eugenol compared to 0.44±0.08mM for lidocaine. The tonic NaV blockade was more effective when the membrane was held at more depolarized, still sublimiar, holding potentials. This observation indicates a higher affinity of eugenol for closed substates dwelled at less hyperpolarized potentials. No consistent evidences for additional binding to open state were observed. A displacement of steady-state inactivation curve to more negative potentials, associated with a slower recovery from fast inactivation under eugenol indicates that this molecule also binds to fast inactivated state. For currents undergoing slow inactivation, a consistent reduction by eugenol indicates that the phenylpropene additionally binds to the slow inactivated state. A frequency-dependent blocking effect of eugenol on NaV was observed, but the effect is smaller than that induced by lidocaine. In conclusion, eugenol binds to several isoforms of TTX-R NaV and to the different states of the proteins, leading to a channel blockage.