Yasunobu Okada

The Puzzles of Volume-Activated Anion Channels

This chapter deals with the anion channels and their multiple functions. Anion channels (ACs) are present both in the plasma membrane and in the membranes of intracellular organelles. Animal cells express a large variety of anion channels in their plasma membrane. ACs are involved in a wide range of functions, such as inhibitory synaptic transmission through plasma membrane hyperpolarization, epithelial Clˉ transport, as well as transport of other organic anions such as glutamate and anionic forms of ATP. In contrast to cation channels, ACs are not directly involved in the initiation and termination of action potentials in nerves and muscles. In neurons and other cell types, membrane potential is determined by the relative electromotive forces and the conductances of each ion permeation pathway.

The organic anion transporter SLCO2A1 constitutes the core component of the Maxi-Cl channel

The maxi‐anion channels (MACs) are expressed in cells from mammals to amphibians with ~60% exhibiting a phenotype called Maxi‐Cl. Maxi‐Cl serves as the most efficient pathway for regulated fluxes of inorganic and organic anions including ATP. However, its molecular entity has long been elusive. By subjecting proteins isolated from bleb membranes rich in Maxi‐Cl activity to LC‐MS/MS combined with targeted siRNA screening, CRISPR/Cas9‐mediated knockout, and heterologous overexpression, we identified the organic anion transporter SLCO2A1, known as a prostaglandin transporter (PGT), as a key component of Maxi‐Cl. Recombinant SLCO2A1 exhibited Maxi‐Cl activity in reconstituted proteoliposomes. When SLCO2A1, but not its two disease‐causing mutants, was heterologously expressed in cells which lack endogenous SLCO2A1 expression and Maxi‐Cl activity, Maxi‐Cl currents became activated. The charge‐neutralized mutant became weakly cation‐selective with exhibiting a smaller single‐channel conductance. Slco2a1 silencing in vitro and in vivo, respectively, suppressed the release of ATP from swollen C127 cells and from Langendorff‐perfused mouse hearts subjected to ischemia–reperfusion. These findings indicate that SLCO2A1 is an essential core component of the ATP‐conductive Maxi‐Cl channel.

Molecular Identities and ATP Release Activities of Two Types of Volume-Regulatory Anion Channels, VSOR and Maxi-Cl

An elaborate volume regulation system based on interplay of ion channels and transporters was evolved to cope with constant osmotic challenges caused by intensive metabolism, transport and other physiological/pathophysiological events. In animal cells, two types of anion channels are directly activated by cell swelling and involved in the regulatory volume decrease (RVD): volume-sensitive outwardly rectifying anion channel (VSOR), also called volume-regulated anion channel (VRAC), and Maxi-Cl which is the most major type of maxi-anion channel (MAC). These two channels have very different biophysical profiles and exhibit opposite dependence on intracellular ATP. After several decades of verifying many false-positive candidates for VSOR and Maxi-Cl, LRRC8 family proteins emerged as major VSOR components, and SLCO2A1 protein as a core of Maxi-Cl. Still, neither of these proteins alone can fully reproduce the native channel phenotypes suggesting existence of missing components. Although both VSOR and Maxi-Cl have pores wide enough to accommodate bulky ATP4- and MgATP2- anions, evidence accumulated hitherto, based on pharmacological and gene silencing experiments, suggests that Maxi-Cl, but not VSOR, serves as one of the major pathways for the release of ATP from swollen and ischemic/hypoxic cells. Relations of VSOR and Maxi-Cl with diseases and their selective pharmacology are the topics promoted by recent advance in molecular identification of the two volume-activated, volume-regulatory anion channels.

Cellular mechanism for herbal medicine Junchoto to facilitate intestinal Cl−/water secretion that involves cAMP-dependent activation of CFTR

Constipation is a common symptom frequently compromising the quality of daily life. Several mechanistically different drugs have been used to mitigate constipation, including Japanese herbal (Kampo) medicines. However, the mechanisms of their actions are often not well understood. Here we aimed to investigate the molecular mechanisms underlying the effects of Junchoto (JCT), a Kampo medicine empirically prescribed for chronic constipation. Cl− channel activity was measured by the patch-clamp method in human cystic fibrosis transmembrane conductance regulator (CFTR)-expressing HEK293T cells and human intestinal Caco-2 cells. cAMP was measured by a luciferase-based assay. Cell volume change was measured by a particle-sizing and particle-counting analyzer and video-microscopic measurement. In both CFTR-expressing HEK293T and Caco-2 cells, JCT dose-dependently induced whole-cell currents showing typical biophysical and pharmacological features of CFTR. Robust expression of CFTR was confirmed by RT-PCR and Western blotting in Caco-2 cells. Luciferase-based measurement revealed that JCT increases intracellular cAMP levels. Administration of the adenylate cyclase inhibitor SQ22536 or CFTR inhibitor-172, or treatment with small interfering RNAs (siRNA) targeting CFTR, abolished JCT-induced whole-cell currents, suggesting that elevated intracellular cAMP by JCT causes activation of CFTR in Caco-2 cells. Finally, blockade of CFTR activity by CFTR inhibitor-172 or siRNA-knockdown of CFTR or application of SQ22536 markedly reduced the degree of cell volume decrease induced by JCT. JCT can induce a Cl− efflux through the CFTR channel to promote water secretion, and this effect is likely mediated by increased cAMP production.