A C-terminally truncated mouse Best3 splice variant targets and alters the ion balance in lysosome-endosome hybrids and the endoplasmic reticulum

Abstract

The Bestrophin family has been characterized as Cl− channels in mammals and Na + channels in bacteria, but their exact physiological roles remian unknown. In this study, a natural C-terminally truncated variant of mouse Bestrophin 3 (Best3V2) expression in myoblasts and muscles is demonstrated. Unlike full-length Best3, Best3V2 targets the two important intracellular Ca stores: the lysosome and the ER. Heterologous overexpression leads to lysosome swelling and renders it less acidic. Best3V2 overexpression also results in compromised Ca 2+ release from the ER. Knocking down endogenous Best3 expression in myoblasts makes these cells more excitable in response to Ca 2+ mobilizing reagents, such as caffeine. We propose that Best3V2 in myoblasts may work as a tuner to control Ca 2+ release from intracellular Ca 2+ stores.

Bestrophin-3 Expression in a Subpopulation of Astrocytes in the Neonatal Brain After Hypoxic-Ischemic Injury

“…The PCR analysis of Best3 mRNA was made by using specifically made primer pairs published previously (Golubinskaya et al, 2015) spanning the expected areas of splicing of exons 2, 3, and 6 (Figure 2A) based on earlier published data on alternative splicing in Best3 mRNA in mouse tissues (Krämer et al, 2004; Srivastava et al, 2008). We did not investigate the possible splicing of exon 10 that recently has been described by Wu et al (2016), so in our PCR experiments the “-2-3+6” and “-2-3-6” splice variants of mRNA could represent corresponding groups of splice variants where also exon 10 is alternatively spliced. Analysis of the ipsilateral, injured hemisphere and the contralateral non-injured hemisphere (Figure 2B–D) showed the presence of two splice variants of Best3 in both hemispheres: one longer variant where exon 6 was present (“+6” variant) and another shorter one, “-6” variant, where exon 6 was spliced out.…”

“…Similar results were obtained in our PCR analysis of Best3 splicing in the brains of neonatal mice (Figure 2), and the composition of splice variants was similar in contralateral non-injured and ipsilateral injured brain hemispheres. However, there is also recent data on possible splicing of the C-terminal region of Best3 mRNA and protein in mouse myoblasts (Wu et al, 2016). If this is the case also in brain, the “+6” and “-6” variants seen in our experiments could represent more than one transcript each.…”

“…Some splice variants of Best3 have the channel pore region spliced out, likely causing them to lose ability to act as ion channels (Srivastava et al, 2008). A specific splice variant has been suggested to control intracellular calcium release in myoblasts, possibly preventing calcium overload (Wu et al, 2016). Best3 has recently also been implicated in various cell-protective mechanisms outside the brain.…”

From Pinocytosis to Methuosis—Fluid Consumption as a Risk Factor for Cell Death

“…In B-cell non-Hodgkin lymphoma cells, the PIKFyfe inhibitor apilimod leads to CD by formation of giant vacuoles and disruption of endolysosomal function, an effect that requires functional ClC-7/Ostm1 transporters ( Gayle et al, 2017 ). In Hela and NIH3T3 cells, a short natural ClC3 splice variant (Clc3s) has been shown to lead to the formation of large vacuoles ( Wu et al, 2016 ), and in Chinese hamster ovary CHO-K1 or human hepatoma Huh-7 cells, the volume of such vesicles is governed by their Cl – concentration ( Li et al, 2002 ).…”

“…The mechanisms leading to swelling-induced rise of pH ves are still elusive. Interestingly, overexpression of a naturally occurring C-terminally truncated splice variant of mouse bestrophin-3, Best3V2, leads to, besides swelling, alkalinization of lysosomes ( Wu et al, 2016 ). Manipulation of the intracellular Cl – concentration leads to alterations of lysosomal volume due to the high ClC-3-endowed Cl – permeability.…”