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  • br Roles for intracellular chloride channels br

    2020-02-19


    Roles for intracellular chloride channels
    Specific chloride channel proteins in intracellular membranes
    Concluding remarks
    Introduction The sulfonylurea derivative glybenclamide (GLYB) is used as oral hypoglycemic agent to treat non-insulin dependent diabetes mellitus [1], [2]. The antidiabetic sulfonylureas in general bind to high affinity sulfonylurea receptors, which are the structural elements of ATP-sensitive potassium (KATP) channels, and so inhibit the channels [3], [4]. The property of GLYB to inhibit KATP channel is widely used as a pharmacological tool in numerous studies including mitochondria [3]. Effect of GLYB on mitochondrial function is complex. It activates cyclosporine A-sensitive mitochondrial permeability transition, induces swelling of mitochondria, increases calcium efflux, inhibits K+ and Na+ uniports, decreases the mitochondrial membrane potential, inhibits respiration and interferes with mitochondrial bioenergetics or reduced intracellular ATP level [4], [5], [6], [7], [8]. The complexity of the GLYB effects indicates that more than one molecular mechanism is involved. Since it was observed that GLYB inhibited cystic fibrosis transmembrane regulator (CFTR), swelling-activated, and Ca2+-activated Cl− channels in cardiac myocytes and plasma membrane of cultured adenosine deaminase [9], [10], [11] it was of interest to know whether GLYB interacts with mitochondrial chloride channels what might contribute to understand its complex mitochondrial effect.
    Material and methods
    Results
    Discussion The single channel properties of the observed mitochondrial chloride channels were different from reported voltage dependent anion channel (VDAC) of the mitochondrial outer membrane. The VDAC has conductance 4nS in 1M KCl, ionic selectivity 2:1 for Cl− over K+. The VDAC closes to subconductance states when either positive or negative potentials are applied [14]. On contrary, we measured chloride channels with lower conductance, higher selectivity for Cl− over K+ and channels did not close to subconductance states when either positive or negative voltage was applied. Our observed chloride channels share some characteristics with the centum pS channels like the response to ATP or conductance [12], [15]. It is now recognized that activation of mitochondrial KATP channels in cardiac myocytes is an important and potent cardioprotective mechanism [16], [17], [18]. However, recently it was proposed that chloride channels also contribute to ischemic preconditioning in the myocardium [19], [20]. The effect of ischemic preconditioning was abolished in CFTR knock-out mice [20]. It has been proposed that anion efflux through mitochondrial inner membrane anion channel (IMAC) may be a safeguard against excessive matrix swelling. Mitochondrial anion efflux has also recently been implicated in regulation of mitochondrial membrane potential. Inhibition of chloride flux by 4′-chlorodiazepam is beneficial for the recovery from ischemia/reperfusion process and it prevents from the collapse of mitochondrial membrane potential during oxidative stress [21], [22]. GLYB is a sulfonylurea drug that is widely used to treatment of diabetes mellitus of type 2 [23]. At nanomolar concentrations it binds to the sulfonylurea receptor in the plasma membrane of pancreatic β-cells to cause the inhibition of KATP channels and promote insulin secretion [24]. Higher (μM) concentrations of GLYB inhibit KATP channels in other tissues [25], including skeletal muscle [26] or rat ventricular myocytes [27]. GLYB blocks pancreatic β-cells KATP and cardiac KATP channels with high affinity; but only for cardiac KATP channels was this block reversible [28]. The sulfonylureas can bind to various intracellular organelles including mitochondria [3]. Its effect on mitochondrial KATP channel have been observed both in patched mitochondria [29] and in proteoliposomes with partly purified mitochonodrial KATP channel reconstituted into bilayer lipid membrane [29], [30]. This channel is blocked by GLYB [30], [31] with a IC50 value below 100nM [31].