The resistance of the inner vestibule limits Shaker’s conductance.

Neurons encode electrical signals with critically tuned voltage-gated ion channels and enzymes. Dedicated voltage sensor domains (VSDs) in these membrane proteins activate coordinately with an unresolved structural change. Such change conveys the transmembrane translocation of four positively charged arginine side chains, the voltage-sensing residues (VSRs; R1-R4). Countercharges and lipid phosphohead groups likely stabilize these VSRs within the low-dielectric core of the protein. However, the role of hydration, a sign-independent charge stabilizer, remains unclear. We replaced all VSRs and their neighboring residues with negatively charged aspartates in a voltage-gated potassium channel. The ensuing mild functional effects indicate that hydration is also important in VSR stabilization. The voltage dependency of the VSR aspartate variants approached the expected arithmetic summation of charges at VSR positions, as if negative and positive side chains faced similar pathways. In contrast, aspartates introduced between R2 and R3 did not affect voltage dependence as if the side chains moved outside the electric field or together with it, undergoing a large displacement and volumetric remodeling. Accordingly, VSR performed osmotic work at both internal and external aqueous interfaces. Individual VSR contributions to volumetric works approached arithmetical additivity but were largely dissimilar. While R1 and R4 displaced small volumes, R2 and R3 volumetric works were massive and vectorially opposed, favoring large aqueous remodeling during VSD activation. These diverse volumetric works are, at least for R2 and R3, not compatible with VSR translocation across a unique stationary charge transfer center. Instead, VSRs may follow separated pathways across a fluctuating low-dielectric septum.

β‐Subunits of the Ca2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore‐forming α1 subunit. However, they comprise protein–protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the β2a subunit of the Ca2+ channels (CaVβ2a) and its SH3 domain (β2a‐SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaVβ2a or β2a‐SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole‐cell patch clamp recordings, Ca2+ currents were reduced by CaVβ2a in the presence of exogenous α1‐interaction domain. β2a‐SH3, but not its dimerization‐deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following β2a‐SH3 injection. Furthermore, β2a‐SH3 was still able to reduce Ca2+ currents when dynamin‐2 function was disrupted, but not when SNARE‐dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both CaVβ2a and β2a‐SH3 diminished the incorporation of new actin monomers to cortical actin filaments, β2a‐SH3 emerges as a signaling module that might down‐regulate forward trafficking of ion channels by modulating actin dynamics.

channels which dramatically impairs stable recordings and thus reliable pharmacological assessment, especially in the study of slow or use-dependent compounds. Nanion's SyncroPatch 384PE offers a high throughput gigaseal platform which records from up to 384/768 experiments at the same time. It does not only allow for high quality recordings with reliable pharmacology, but also for a more detailed biophyiscal characterization of an ion channel protein. A temperature control feature supports measurements at physiological temperatures. Our results show very stable recordings of CaV1.2 over an extended period of time (> 25 min.) that permits for cumulative compound application. Furthermore, we demonstrate CaV1.2 activation from different states that discriminates state-and use-dependent drug effects. Our assay development demonstrates how accurate pharmacology and high-throughput recordings of even difficult targets like CaV1.2 can be achieved in a reproducible and reliable manner with the SyncroPatch 384PE. 2194-Pos Board B338Eminence of VSD I in the Voltage-Dependent Inactivation of the Human Ca V 1.2 Channel