Biology subjects

Flucher, B. E.

Publications and source records attributed to Flucher, B. E..

3 recordsLinked to original sources

bioRxiv2025

A bi-directional binding site linking the α2δ-1 subunit to the intrinsic speed control process in VSD I of voltage-gated calcium channels

Voltage-gated calcium channels communicate electrical signals in membranes of excitable cells into cellular responses like secretion of hormones and neurotransmitters, or the contraction of heart and skeletal muscle cells. Their activation properties are tuned to match their specific functions. Consequently, the different members of the calcium channel family activate over a wide range of voltages and with greatly differing speeds. The skeletal muscle CaV1.1 and the cardiac/neuronal CaV1.2 represent two structurally closely related channels with particularly slow and fast activation kinetics, respectively. Both channel paralogs associate with the auxiliary calcium channel subunit 2{delta}-1, which is a known regulator of activation properties. By expressing CaV1.1 and CaV1.2 with and without 2{delta}-1 in a new double-knockout muscle cell line, we demonstrate that 2{delta}-1 regulates activation kinetics of the two channels in opposite directions. Molecular dynamics simulation revealed a string of charged amino acids connecting 2{delta}-1 to the intrinsic speed-control mechanism of voltage-sensing domain I (VSD I) in CaV1.1. Charge-neutralizing mutations of any of these charged amino acids abolished the 2{delta}-1 modulation and accelerated current kinetics. Together, these results reveal the molecular mechanism by which the 2{delta}-1 subunit regulates the intrinsic speed-control mechanism in the VSD I of CaV1.1 calcium channels.

biophysics↗

bioRxiv2023

Asymmetric Contribution of a Selectivity Filter Gate in Triggering Inactivation of CaV1.3 Channels

Voltage-dependent and Ca2+-dependent inactivation (VDI and CDI, respectively) of CaV channels are two biologically consequential feedback mechanisms that fine-tune Ca2+ entry into neurons and cardiomyocytes. Although known to be initiated by distinct molecular events, how these processes obstruct conduction through the channel pore remains poorly defined. Here, focusing on ultra-highly conserved tryptophan residues in the inter-domain interfaces near the selectivity filter of CaV1.3, we demonstrate a critical role for asymmetric conformational changes in mediating VDI and CDI. Specifically, mutagenesis of the domain III-IV interface, but not others, enhanced VDI. Molecular dynamics simulations demonstrate that mutations in distinct selectivity filter interfaces differentially impact conformational flexibility. Furthermore, mutations in distinct domains preferentially disrupt CDI mediated by the N- versus C-lobes of CaM, thus uncovering a scheme of structural bifurcation of CaM signaling. These findings highlight the fundamental importance of the asymmetric arrangement of the pseudo-tetrameric CaV pore domain for feedback inhibition.

biophysics↗

bioRxiv2021

Calcium current modulation by the γ1 subunit depends on alternative splicing of CaV1.1

The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as voltage sensor for excitation-contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming 1S subunit and the auxiliary 2{delta}-1 and {gamma}1 subunits. Particularly, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage-dependence of activation to positive potentials outside the physiological range. We generated a new HEK293-cell line stably expressing 2{delta}-1, {beta}3, and STAC3. When the adult (CaV1.1a) and the embryonic (CaV1.1e) splice variants were expressed in these cells, the difference in the voltage-dependence of activation observed in muscle cells was reproduced, but not the reduced current density of CaV1.1a. Only when we further co-expressed the {gamma}1 subunit, the current density of CaV1.1a, but not of CaV1.1e, was reduced by >50 %. In addition, {gamma}1 caused a shift of the voltage-dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of {gamma}1, but not its effect on inactivation, is specifically dependent on the inclusion of exon 29 in CaV1.1a. Molecular structure modeling revealed several direct ionic interactions between oppositely charged residues in the IVS3-S4 loop and the {gamma}1 subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the {gamma}1-dependent reduction of current density, suggesting that structural rearrangements of CaV1.1a induced by inclusion of exon 29 allosterically empower the {gamma}1 subunit to exert its inhibitory action on CaV1.1 calcium currents. SummaryEl Ghaleb et al. analyzed the effects of the {gamma}1 subunit on current properties and expression of the adult (CaV1.1a) and embryonic (CaV1.1e) calcium channel splice variants, demonstrating that {gamma}1 reduces the current amplitude in a splicing-dependent manner.

physiology↗