In the nervous system, A→I RNA editing has an important role in regulating neuronal excitability. Ligand-gated membrane receptors, synaptic proteins, as well as ion channels, are targets for recoding by RNA editing. Although scores of editing sites have been identified in the mammalian brain, little is known about the functional alterations that they cause, and even less about the mechanistic underpinnings of how they change protein function. We have previously shown that an RNA editing event (I400 V) alters the inner permeation pathway of human KV1.1, modifying the kinetics of fast inactivation. Here we show that the channel's inactivation gate enters deep into the ion permeation pathway and the very tip establishes a direct hydrophobic interaction with the edited position. By converting I to V, the intimacy of the interaction is reduced, allowing the inactivation gate to unbind with much faster kinetics.

Calcium-activated chloride channels (CaCC) formed by anoctamin1/TMEM16A subunits are ubiquitously expressed, and these channels are known to prevent polyspermy in amphibian oocytes. Here, we describe a TMEM16A clone isolated from Xenopus tropicalis oocytes (xtTMEM16A) and how the anion permeation properties are modified in single-site mutants of the ion pore. The anion permeability sequence was SCN(-) > I(-) > Br(-) > Cl(-) > gluconate (relative permeabilities 5.6:3.0:2.1:1:0.2, respectively). Dose-response curves indicated that the voltage-dependent half-maximal concentration for Ca(2+) activation (K d of the Hill equation at +100 mV) was 120 nM in normal external Cl(-), whereas it was displaced leftward to 75 nM Ca(2+), when I(-) replaced Cl(-). The I(-):Cl(-) mole fraction (MF) of the external solution was varied in order to gain insight into the permeation mechanism of the pore. No anomaly in MF behavior was observed for conductance, but it was observed for current reversal potential, which deviated from the prediction of the Goldman-Hodgkin-Katz equation. Mutations of positively charged amino acids in the pore, R646 and R761, to glutamate resulted in reduction of the relative permeability to I(-). Data from the wild type and mutants could be well fitted by a three-barrier, two-site permeation model. This suggests a multi-ion pore with at least two binding sites for anions, with R646 mole fraction closer to the extracellular membrane surface--being important for the stability of both sites--and R761--located deeper within the membrane--mainly affecting the innermost binding site. Considerations of xtTMEM16A putative pore region topology are discussed in the light of two alternative topological models of the protein.

Significance Immunosuppression by myeloid-derived suppressor cells (MDSC), especially near tumor surfaces, involves the extracellular production of reactive oxygen species (ROS). ROS generation in MDSC occurs during the oxidation of NADPH to NADP+, which NOX2 catalyzes. ROS react with the T cell receptor complex, abolishing the antigen presentation, which blocks the immune system elimination of the tumor cells. Extrusion of protons from MDSC by voltage-gated proton channel (H v 1) sustains ROS production. Here, we demonstrate the expression of H v 1 in mouse MDSC. In this way, H v 1 present in MDSC becomes a potential cancer therapeutic target since its inhibition seems to diminish immunosuppression activity in the tumoral microenvironment, allowing cancer cells to be attacked by the immune system.