Biochimica et Biophysica Acta 806 (1985) 195-209 195 Elsevier BBA 41680 Respiration induces variable porosity to polyols in the mitochondrial inner membrane D. Sambasivarao and V. Sitaramam National Institute of Nutrition, Indian Council of Medical Research, Jamai Osmania P. 0., Hyderabad-500 007, A.P. (India) (Received July 25th, 1984) Key words: Mitochondrial swelling; Enzyme osmometry; Respiration; Oxidative phosphorylation; Membrane porosity; (Rat liver) The osmotic basis of low and high amplitude swelling in mitochondria was investigated in detail using sucrose and mannitoi as external osmolytes. Osmotic behaviour of mitochondria in various respiratory states was consistent with significant changes in the porosity of the inner membrane corresponding to the rate of respiration. The stoichiometry of oxidative phosphorylation was confirmed to he dependent on the physical state (i.e., osmotic stretch) of the inner membrane regardless of the external polyol used. High amplitude swelling in polyol media was shown to arise from a sequential disruption of the outer and inner mitochondrial membranes, due to a dynamic instability induced by a combination of respiration, unscreened (fixed) surface charge density and the consequent variable porosity of the inner membrane. These novel experimental findings based on the physical theory of osmosis emphasize the need to define the fine structural changes of the inner membrane associated with oxidative phosphorylation to arrive at a comprehensive mechanism. Introduction The marked nonadiabatic behaviour of mitochondria, as manifest by relatively rapid con- traction-swelling cycles in isotonic media associ- ated with oxidative phosphorylation (low ampli- tude swelling)or due to respiration in nonelectro- lyte media (high amplitude swelling) [1,2], remains a major unsolved paradox. The paradox lies in that, swelling (and/or contraction) and an in- variant isotonicity (i.e., invariant permeability characteristics of the inner membrane) cannot coexist in a membrane-bound organelle that other- wise behaves as a perfect osmometer [3]. The paradox becomes particularly acute in the light of the chemiosmotic hypothesis of Mitchell [4]. Since proton translocations are conceived to be media- ted by specific pumps embedded in a membrane with otherwise low proton conductance, presence of even a single gramicidin channel (pore radius --- 0.4 nm [5]) would discharge the postulated proton gradient [6]! Since the external osmolyte in mitochondrial studies is usually sucrose (0.53 nm radius [7]), the phenomenon of swelling can occur only if pores of larger radius are induced in nor- mal mitochondria, leading to colloidal swelling (cf. Ref. 8). Can proton motive force exist across a membrane with large porosity? This paradox closely parallels and is a historical consequence of yet another riddle which resulted in sucrose space hypothesis [.9,10]. The paradoxical coexistence of rapid entry kinetics of sucrose dur- ing centrifugation into subcellular organdies [11], notably the mitochondria, which otherwise behave as perfect osmometers [3] led to the postulation of a two-compartment model, in which the intermem- branous space was designated as the sucrose-per- meable space and the matrix space as the sucrose- 0005-2728/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)