Biology subjects

Bassetto, C. A. Z.

Publications and source records attributed to Bassetto, C. A. Z..

4 recordsLinked to original sources

bioRxiv2024

Turning a Kv channel into hot and cold receptor by perturbing its electromechanical coupling.

Voltage-dependent potassium channels (Kv) are extremely sensitive to membrane voltage and play a crucial role in membrane repolarization during action potentials. Kv channels undergo voltage-dependent transitions between closed states before opening. Despite all we have learned using electrophysiological methods and structural studies, we still lack a detailed picture of the energetics of the activation process. We show here that even a single mutation can drastically modify the temperature response of the Shaker Kv channel. Using rapid cell membrane temperature steps (Tsteps), we explored the effects of temperature on the ILT mutant (V369I, I372L, and S376T) and the I384N mutant. The ILT mutant produces a significant separation between the transitions of the voltage sensor domain (VSD) activation and the I384N uncouples its movement from the opening of the domain (PD). ILT and I384N respond to temperature in drastically different ways. In ILT, temperature facilitates the opening of the channel akin to a "hot" receptor, reflecting the temperature dependence of the voltage sensors last transition and facilitating VSD to PD coupling (electromechanical coupling). In I384N, temperature stabilizes the channel closed configuration analogous to a "cold" receptor. Since I384N drastically uncouples the VSD from the pore opening, we reveal the intrinsic temperature dependence of the PD itself. Here, we propose that the electromechanical coupling has either a "loose" or "tight" conformation. In the loose conformation, the movement of the VSD is necessary but not sufficient to efficiently propagate the electromechanical energy to the S6 gate. In the tight conformation the VSD activation is more effectively translated into the opening of the PD. This conformational switch can be tuned by temperature and modifications of the S4 and S4-S5 linker. Our results show that we can modulate the temperature dependence of Kv channels by affecting its electromechanical coupling.

biophysics↗

bioRxiv2023

Photolipid excitation triggers depolarizing optocapacitive currents and action potentials

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, we 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. HighlightsO_LIRapid photolipid photoisomerization generates optocapacitive currents in planar lipid bilayers and HEK293 cells. C_LIO_LIThese currents originate from photo-induced changes in membrane capacitance C_LIO_LIUV light-triggered membrane depolarization opens NaV1.3, evoking action potentials. C_LIO_LIBlue light-induced mechanosensitive channel opening gives rise to depolarizing currents, which may evoke NaV1.3-mediated action potentials. C_LI

biophysics↗

bioRxiv2022

ION CHANNEL THERMODYNAMICS STUDIED WITH TEMPERATURE JUMPS MEASURED AT THE MEMBRANE

Perturbing the temperature of a system modifies its energy landscape thus providing a ubiquitous tool to understand biological processes. Here, we developed a framework to generate sudden temperature jumps (Tjumps) and sustained temperature steps (Tsteps) to study the temperature dependence of membrane proteins under voltage-clamp, while measuring the membrane temperature. Utilizing the melanin under the Xenopus laevis oocytes membrane as a photothermal transducer, we achieved short Tjumps up to 10 {o}C in less than 1.5 ms and constant Tsteps for durations up to 150 ms. We followed the temperature at the membrane with submillisecond time resolution by measuring the time-course of membrane capacitance, which is linearly related to temperature. We applied Tjumps in Kir 1.1b, which reveals a highly temperature-sensitive blockage relief and characterized the effects of Tsteps on the temperature-sensitive channels TRPM8 and TRPV1. These newly developed approaches provide a general tool to study membrane proteins thermodynamics.

biophysics↗

bioRxiv2019

Metal bridge in S4 segment supports helix transition in Shaker channel

Voltage-gated ion channels play important roles in physiological processes, especially in excitable cells, where they shape the action potential. In S4-based voltage sensors voltage-gated channels, a common feature is shared: the transmembrane segment 4 (S4) contains positively charged residues intercalated by hydrophobic residues. Although several advances have been made in understating how S4 moves through a hydrophobic plug upon voltage changes, possible helix transition from -to 310-helix in S4 during activation process is still unresolved. Here, we have mutated several hydrophobic residues from I360 to F370 in the S4 segment into histidine, in i, i+3 and i, i+6 or i, i+4 and i, i+7 pairs, to favor 310- or -helical conformations, respectively. We have taken advantage that His can be coordinated by Zn+2 to promote metal ion bridges and we have found that the histidine introduced at position 366 (L366H) can interact with the introduced histidine at position 370 (stabilizing that portion of the S4 segment in -helical conformation). In presence of 20 M of Zn+2, the activation currents of L366H:F370H channels were slowed down by a factor of 3.5, the voltage-dependence is shifted by 10 mV towards depolarized potentials with no change on the deactivation time constant. Our data supports that by stabilizing a region of the S4 segment in -helical conformation a closed (resting or intermediate) state is stabilized rather than destabilizing the open (active) state. Taken together, our data indicates that the S4 undergoes -helical conformation to a short-lived different secondary structure transiently before reaching the active state in the activation process.\n\nSTATEMENT OF SIGNIFICANCEConformational transitions between -helix and 310-helix in the S4 segment of Shaker potassium channel during gating has been under debate. The present study shows the coordination by Zn2+ of a pair of engineered histidine residues (L366H:F370H) in the intermediate region of S4 in Shaker, favoring -helical conformation. In presence of 20M of Zn+2 the activation currents of L366H:F370H channels become slower, with 10 mV positive shift in the voltage-dependence and no effects on deactivation time constants suggesting a stabilization of a closed state rather than destabilization the open (active) state. Collectively, our data indicate that S4 undergoes secondary structure changes, including a short-lived secondary structure transition, when S4 moves from the resting to the active state during activation.

biophysics↗