Dimerization is required for the glycosylation of S1-S2 linker of sea urchin voltage-gated proton channel Hv1.

Multimerization of ion channels is essential for establishing the ion-selective pathway and tuning the gating regulated by membrane potential, second messengers, and temperature. Voltage-gated proton channel, Hv1, consists of voltage-sensor domain and coiled-coil domain. Hv1 forms dimer, whereas voltage-dependent channel activity is self-contained in monomer unlike many ion channels, which assemble to form ion-conductive pathways among multiple subunits. Dimerization of Hv1 is necessary for cooperative gating, but other roles of dimerization in physiological aspects are still largely unclear. In this study, we show that dimerization of Hv1 takes place in ER. Sea urchin Hv1 (Strongylocentrotus purpuratus Hv1: SpHv1) was glycosylated in the consensus sequence for N-linked glycosylation within the S1-S2 extracellular loop. However, glycosylation was not observed in the monomeric SpHv1 that lacks the coiled-coil domain. A version of mHv1 in which the S1-S2 loop was replaced by that of SpHv1 showed glycosylation and its monomeric form was not glycosylated. Tandem dimer of monomeric SpHv1 underwent glycosylation, suggesting that dimerization of Hv1 is required for glycosylation. Moreover, when monomeric Hv1 has a dilysine motif in the C-terminal end, which is known to act as a retrieval signal from Golgi to ER, prolonging the time of residency in ER, it was glycosylated. Overall, our results suggest that monomeric SpHv1 does not stay long in ER, thereby escaping glycosylation, while the dimerization causes the proteins to stay longer in ER. Thus, the findings highlight the novel significance of dimerization of Hv1: regulation of biogenesis and maturation of the proteins in intracellular compartments.

Insight into the function of voltage-sensing phosphatase in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni.

Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.

Structural change of the cytoplasmic N-terminus and S1 segment of voltage-sensing phosphatase reported by Anap.

BACKGROUND: Voltage-sensing phosphatase contains a structurally conserved S1-S4-based voltage-sensor domain, which undergoes a conformational transition in response to membrane potential change. Unlike that of channels, it is functional even in isolation and is therefore advantageous for studying the transition mechanism, but its nature has not yet been fully elucidated. This study aimed to address whether the cytoplasmic N-terminus and S1 exhibit structural change. METHODS: Anap, an environment-sensitive unnatural fluorescent amino acid, was site-specifically introduced to the voltage sensor domain to probe local structural changes by using oocyte voltage clamp and photometry. Tetramethylrhodamine was also used to probe some extracellularly accessible positions. In total, 51 positions were investigated. RESULTS: We detected robust voltage-dependent signals from widely distributed positions including N-terminus and S1. In addition, response to hyperpolarization was observed at the extracellular end of S1, reflecting the local structure flexibility of the voltage-sensor domain in the down-state. We also found that the mechanical coupling between the voltage-sensor and phosphatase domains affects the depolarization-induced optical signals but not the hyperpolarization-induced signals. CONCLUSIONS: These results fill a gap between the previous interpretations from the structural and biophysical approaches and should provide important insights into the mechanisms of the voltage-sensor domain transition as well as its coupling with the effector.

(R)-ketamine restores anterior insular cortex activity and cognitive deficits in social isolation-reared mice.

Chronic social isolation increases the risk of mental health problems, including cognitive impairments and depression. While subanesthetic ketamine is considered effective for cognitive impairments in patients with depression, the neural mechanisms underlying its effects are not well understood. Here we identified unique activation of the anterior insular cortex (aIC) as a characteristic feature in brain-wide regions of mice reared in social isolation and treated with (R)-ketamine, a ketamine enantiomer. Using fiber photometry recording on freely moving mice, we found that social isolation attenuates aIC neuronal activation upon social contact and that (R)-ketamine, but not (S)-ketamine, is able to counteracts this reduction. (R)-ketamine facilitated social cognition in social isolation-reared mice during the social memory test. aIC inactivation offset the effect of (R)-ketamine on social memory. Our results suggest that (R)-ketamine has promising potential as an effective intervention for social cognitive deficits by restoring aIC function.