Higher expression of KCNK10 (TREK-2) K+ channels and their functional upregulation by lipopolysaccharide treatment in mouse peritoneal B1a cells.

Innate-like CD5+ B1a cells localized in serous cavities are activated by innate stimuli, such as lipopolysaccharide (LPS), leading to T cell-independent antibody responses. Although ion channels play crucial roles in the homeostasis and activation of immune cells, the electrophysiological properties of B1a cells have not been investigated to date. Previously, in the mouse B cell lymphoma cells, we found that the voltage-independent two-pore-domain potassium (K2P) channels generate a negative membrane potential and drive Ca2+ influx. Here, we newly compared the expression and activities of K2P channels in mouse splenic follicular B (FoB), marginal zone B (MZB), and peritoneal B1a cells. Next-generation sequencing analysis showed higher levels of transcripts for TREK-2 and TWIK-2 in B1a cells than those in FoB or MZB cells. Electrophysiological analysis, using patch clamp technique, revealed higher activity of TREK-2 with the characteristic large unitary conductance (~ 250 pS) in B1a than that in FoB or MZB cells. TREK-2 activity was further increased by LPS treatment (>2 h), which was more prominent in B1a than that in MZB or FoB cells. The cytosolic Ca2+ concentration of B cells was decreased by high-K+-induced depolarization (ΔRKCl (%)), suggesting the basal Ca2+ influx to be driven by negative membrane potential. The LPS treatment significantly increased the ΔRKCl (%) in B1a, though not in FoB and MZB cells. Our study was the first to compare the K2P channels in mouse primary B cell subsets, elucidating the functional upregulation of TREK-2 and augmentation of Ca2+ influx by the stimulation of Toll-like receptor 4 in B1a cells.

Suppression of hERG K+ current and cardiac action potential prolongation by 4-hydroxynonenal via dual mechanisms.

Oxidative stress under pathological conditions, such as ischemia/reperfusion and inflammation, results in the production of various reactive chemicals. Of these chemicals, 4-hydroxynonenal (4-HNE), a peroxidation product of ω6-polyunsaturated fatty acid, has garnered significant attention. However, the effect of 4-HNE on cardiac electrophysiology has not yet been reported. In the present study, we investigated the effects of 4-HNE on several cardiac ion channels, including human ether-a-go-go-related (hERG) channels, using the whole-cell patch clamp technique. Short-term exposure to 100 µM 4-HNE (4-HNE100S), which mimics local levels under oxidative stress, decreased the amplitudes of rapidly activating delayed rectifier K+ current (IKr) in guinea pig ventricular myocytes (GPVMs) and HEK293T cells overexpressing hERG (IhERG). MS analysis revealed the formation of 4-HNE-hERG adduct on specific amino acid residues, including C276, K595, H70, and H687. Long-term treatment (1-3 h) with 10 µM 4-HNE (4-HNE10L), suppressed IKr and IhERG, but not IKs and ICa,L. Action potential duration (APD) of GPVMs was prolonged by 37% and 64% by 4-HNE100S and 4-HNE10L, respectively. Western blot analysis using surface biotinylation revealed a reduction in mature membrane hERG protein after treatment with 4-HNE10L. Proteasomal degradation inhibitors, such as bortezomib, prevented the 4-HNE10L-induced decrease in mature hERG, suggesting a retrograde degradation of membrane hERG due to 4-HNE. Taken together, 4-HNE100S and 4-HNE10L suppressed IhERG via functional inhibition and downregulation of membrane expression of hERG, respectively. The exposure of 4-HNE under pathological oxidative stress may increase the risk of proarrhythmic events via APD prolongation.