Reciprocal effects between spermine and Mg 2þ on their movements across the mitochondrial membrane Mauro Salvi and Antonio Toninello * Dipartimento di Chimica Biologica, Istituto di Neuroscienze del C.N.R., UniversitaÕ di Padova, UnitaÕ per lo Studio delle Biomembrane, Via G. Colombo 3, 35121 Padova, Italy Received 30 October 2002, and in revised form 16 December 2002 Abstract Mg 2þ competitively inhibits spermine transport in energized rat liver mitochondria (RLM) and exhibits a K i of 0.1 mM on the initial rate and an I 50 of 0.6 mM on total spermine accumulation after 20 min. Addition of 2 mM Mg 2þ after spermine accumulation induces release of the polyamine. In view of the fact that spermine cycles across the inner membrane under physiological conditions, these results demonstrate that Mg 2þ inhibits spermine influx but does not affect the efflux pathway of the polyamine; the inhibitory effect occurs via an interaction with the specific site responsible for spermine transport. Instead, spermine inhibits Mg 2þ binding without affecting the rate of Mg 2þ transport, suggesting that both cations bind to the same site, which, however, is not used for Mg 2þ transport. Spermine also inhibits Mg 2þ efflux from RLM induced under conditions of the ‘‘low conductance state,’’ a pre- liminary step preceding permeability transition pore opening. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Spermine; Mg 2þ ; Mitochondria The polyamine spermine is transported into the ma- trix of RLM 1 by a specific uniporter exhibiting strict DW dependence (for a review on polyamine transport, see [1]). Due to this activity, spermine is normally present in the mitochondrial matrix [2]. Mitochondrial membranes possess two spermine binding sites, both with low-af- finity/high binding capacity [3]. The binding of spermine to one of these sites, called S 1 , represents the pre- liminary step for spermine transport into the matrix and for its inhibitory effect on the mitochondrial perme- ability transition (MPT) [4]. The role of the other site, S 2 , has not yet been well defined, but is probably linked to other effects of spermine on mitochondria [3]. Flux– voltage analyses [4] demonstrated that the spermine transporter is a saturable channel having an asymmet- rical energy profile composed of two barriers, with binding site S 1 located in the energy well between the two peaks [5]. The mechanisms and pathways for Mg 2þ influx and efflux in RLM are not well defined. There is some evidence that uptake takes place by a respiration-de- pendent mechanism [6], through a nonspecific diffusive pathway in response to high DW [7], with saturation kinetics [6]. It has also been proposed that Mg 2þ is transported by a specific pore that differs from other cation transporters [8]. Endogenous Mg 2þ is present mainly in the intermembrane compartment (50%) and the matrix space (41%). Both the outer and the inner membranes contain small amounts of the cation (about 5%). It has been reported that all the mito- chondrial compartments contain several Mg 2þ binding sites that possess low affinity/high binding capacity, similar to the spermine binding sites [9]. Both spermine and Mg 2þ are able to protect RLM against the dele- terious effects of the MPT, with the polyamine being more effective in this regard [10]. The present study investigated the possible reciprocal interactions of both Archives of Biochemistry and Biophysics 411 (2003) 262–266 www.elsevier.com/locate/yabbi ABB * Corresponding author. Fax: +39-049-827-6133. E-mail address: antonio.toninello@unipd.it (A. Toninello). 1 Abbreviations used: MPT, mitochondrial permeability transition; RLM, rat liver mitochondria; TPP þ , tetraphenylphosphonium; LCS, low conductance state. 0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(02)00731-2