N-terminal region is responsible for mHv1 channel activity in MDSCs.

Voltage-gated proton channels (Hv1) are important regulators of the immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in mice and have been proposed as a potential therapeutic target to alleviate dysregulated immunosuppression in tumors. However, till date, there is a lack of evidence regarding the functioning of the Hvcn1 and reports on mHv1 isoform diversity in mice and MDSCs. A computational prediction has suggested that the Hvcn1 gene may express up to six transcript variants, three of which are translated into distinct N-terminal isoforms of mHv1: mHv1.1 (269 aa), mHv1.2 (269 + 42 aa), and mHv1.3 (269 + 4 aa). To validate this prediction, we used RT-PCR on total RNA extracted from MDSCs, and the presence of all six predicted mRNA variances was confirmed. Subsequently, the open-reading frames (ORFs) encoding for mHv1 isoforms were cloned and expressed in Xenopus laevis oocytes for proton current recording using a macro-patch voltage clamp. Our findings reveal that all three isoforms are mammalian mHv1 channels, with distinct differences in their activation properties. Specifically, the longest isoform, mHv1.2, displays a right-shifted conductance-voltage (GV) curve and slower opening kinetics, compared to the mid-length isoform, mHv1.3, and the shortest canonical isoform, mHv1.1. While mHv1.3 exhibits a V0.5 similar to that of mHv1.1, mHv1.3 demonstrates significantly slower activation kinetics than mHv1.1. These results suggest that isoform gating efficiency is inversely related to the length of the N-terminal end. To further explore this, we created the truncated mHv1.2 ΔN20 construct by removing the first 20 amino acids from the N-terminus of mHv1.2. This construct displayed intermediate activation properties, with a V0.5 value lying intermediate of mHv1.1 and mHv1.2, and activation kinetics that were faster than that of mHv1.2 but slower than that of mHv1.1. Overall, these findings indicate that alternative splicing of the N-terminal exon in mRNA transcripts encoding mHv1 isoforms is a regulatory mechanism for mHv1 function within MDSCs. While MDSCs have the capability to translate multiple Hv1 isoforms with varying gating properties, the Hvcn1 gene promotes the dominant expression of mHv1.1, which exhibits the most efficient gating among all mHv1 isoforms.

Direct inhibition of CaV2.3 by Gem is dynamin dependent and does not require a direct alfa/beta interaction.

The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel ß subunit (CaVß), and the binding between CaVα1/CaVß appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVß. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVß independent promotion of dynamin-dependent endocytosis.

The Ca2+ channel subunit CaV ß2a-subunit down-regulates voltage-activated ion current densities by disrupting actin-dependent traffic in chromaffin cells.

ß-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 ß2a subunit of the Ca2+ channels (CaV ß2a) and its SH3 domain (ß2a-SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaV ß2a or ß2a-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 CaV ß2a in the presence of exogenous α1-interaction domain. ß2a-SH3, but not its dimerization-deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following ß2a-SH3 injection. Furthermore, ß2a-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 CaV ß2a and ß2a-SH3 diminished the incorporation of new actin monomers to cortical actin filaments, ß2a-SH3 emerges as a signaling module that might down-regulate forward trafficking of ion channels by modulating actin dynamics.

Gating-induced large aqueous volumetric remodeling and aspartate tolerance in the voltage sensor domain of Shaker K+ channels.

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.

Pore dimensions and the role of occupancy in unitary conductance of Shaker K channels.

K channels mediate the selective passage of K(+) across the plasma membrane by means of intimate interactions with ions at the pore selectivity filter located near the external face. Despite high conservation of the selectivity filter, the K(+) transport properties of different K channels vary widely, with the unitary conductance spanning a range of over two orders of magnitude. Mutation of Pro475, a residue located at the cytoplasmic entrance of the pore of the small-intermediate conductance K channel Shaker (Pro475Asp (P475D) or Pro475Gln (P475Q)), increases Shaker's reported ∼ 20-pS conductance by approximately six- and approximately threefold, respectively, without any detectable effect on its selectivity. These findings suggest that the structural determinants underlying the diversity of K channel conductance are distinct from the selectivity filter, making P475D and P475Q excellent probes to identify key determinants of the K channel unitary conductance. By measuring diffusion-limited unitary outward currents after unilateral addition of 2 M sucrose to the internal solution to increase its viscosity, we estimated a pore internal radius of capture of ∼ 0.82 Å for all three Shaker variants (wild type, P475D, and P475Q). This estimate is consistent with the internal entrance of the Kv1.2/2.1 structure if the effective radius of hydrated K(+) is set to ∼ 4 Å. Unilateral exposure to sucrose allowed us to estimate the internal and external access resistances together with that of the inner pore. We determined that Shaker resistance resides mainly in the inner cavity, whereas only ∼ 8% resides in the selectivity filter. To reduce the inner resistance, we introduced additional aspartate residues into the internal vestibule to favor ion occupancy. No aspartate addition raised the maximum unitary conductance, measured at saturating [K(+)], beyond that of P475D, suggesting an ∼ 200-pS conductance ceiling for Shaker. This value is approximately one third of the maximum conductance of the large conductance K (BK) channel (the K channel of highest conductance), reducing the energy gap between their K(+) transport rates to ∼ 1 kT. Thus, although Shaker's pore sustains ion translocation as the BK channel's does, higher energetic costs of ion stabilization or higher friction with the ion's rigid hydration cage in its narrower aqueous cavity may entail higher resistance.