Abstract
Addition of A23187 plus EDTA to energized mitochondria in KCl medium determines a rapid osmotic swelling due to K+ uptake. The swelling is fully reversed by uncoupler, is stimulated by quinine, and is accompanied by membrane depolarization and increased rate of respiration. A23187-treated mitochondria passively swell in K+ thiocyanate at neutral pH, under conditions where the H+-K+ antiporter appears to be silent. These data indicate that A23187 activates electrophoretic K+ flux, supporting the notion that Mg2+ depletion unmasks several ionic conductance pathways whose concerted interplay could provide a sensitive regulation of mitochondrial volume homeostasis.
Highlights
These controversies were apparently settled by a study of Dordick et al [8], who showed that addition ofA23187 to energized mitochondria incubated inlow K+ medium induced
The problem arises as to debate most authors seem to agree that (i)A23187 treatment unmasks the endogenous, electroneutral H+-K' antiporter as originally proposed by Garlid's group [7, 8]; (ii) antiporter activity is independently stimulated by alkaline pH and osmotic swelling of the matrix [9]; and (iii) uniport K' transport does not play a major role in how the attainmentof an equilibrium distribution for K+ can the observed K+ fluxes
A23187 is a carboxylic acid antibiotic introduced and charmedia (a technique firstintroduced by Chappell and Haarhoff [14] and widely used by a number of authors inkey studies of mitochondrial ion and metabolite transport [2, 14,15,16,17,18,19,20]) or in respiring mitochondria incubated in low K+ media [7]
Summary
Severe depletion of intramitochondrial Mg2+and Ca2+,due to electroneutral H+-Me2+exchange mediated by the ionophore [6]. Preparation of mitochondria, measurements of oxygen consumption, and determination of net K' fluxes with a K+-selectiveelectrode were performed exactly as described previously [9].Membrane potential was monitored with a TPMP+'-selective electrode as described [21].The kinetics of light scattering changes, reflecting mitochondrial volume changes linked to solute transport [14],were measured at 540 nm with eitheraPerkin-Elmer h 5 spectrophotometer or with a Hewlett-Packard 8452A diode array spectrophotometer. 'The abbreviations used are: TPMP+, triphenylmethylphosphonium ion; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Mops, 4-morpholinepropanesulfonicacid.
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