Voltage-dependent Inwardly Rectifying Potassium Conductance in the Outer Membrane of Neuronal Mitochondria

Abstract

Potassium fluxes integrate mitochondria into cellular activities, controlling their volume homeostasis and structural integrity in many pathophysiological mechanisms. The outer mitochondrial membrane (OMM) is thought to play a passive role in this process because K ؉ is believed to equilibrate freely between the cytosol and mitochondrial intermembrane space. By patch clamping mitochondria isolated from the central nervous systems of adult mitoCFP transgenic mice, we discovered the existence of I OMMKi , a novel voltage-dependent inwardly rectifying K ؉ conductance located in the OMM. I OMMKi is regulated by osmolarity, potentiated by cAMP, and activated at physiological negative potentials, allowing K ؉ to enter the mitochondrial intermembrane space in a controlled regulated fashion. The identification of I OMMKi in the OMM supports the notion that a membrane potential could exist across this membrane in vivo and suggests that the OMM possesses regulated pathways for K ؉ uptake.Potassium accumulation in mitochondria is known to be regulated by a uniport mechanism that controls mitochondrial volume homeostasis and bioenergetics (1) and works concurrently with a K ϩ -H ϩ exchanger to prevent mitochondrial swelling (2, 3). In particular, K ϩ entry into mitochondria has been credited to its passive diffusion through the outer mitochondrial membrane (OMM) 5 into the intermembrane compartment and to the K ϩ uniport mechanism, which comprises distinct channels located in the inner mitochondrial membrane (IMM) that allow K ϩ to flow into the matrix (4). In contrast to the vast literature on K ϩ currents through the IMM (4 -6), there have been only a few early attempts to study the electrophysiological properties of the OMM in situ (i.e. in intact isolated mitochondria), which fell short of giving a clear picture of K ϩ across this membrane (7). For instance, the large voltagedependent anion channel (VDAC) of the outer membrane was thoroughly described in reconstituted membrane lipid bilayers (8), but it has never been observed or its biophysical features described in patch-clamp experiments of intact mitochondria. Rather, a variety of conductances in the range of 10 -307 picosiemens (in 150 KCl) have been reported (9, 10), whose function and regulation still await to be assigned (5).To provide an additional contribution to the existing database on OMM conductances, we applied the patch-clamp technique to intact mitochondria isolated from the central nervous system of adult mice. This is a severe technical challenge mainly because of the small size of the organelle (ϳ1 m in diameter) and the difficulty in identifying the single mitochondrion in isolation. We circumvented these problems by using tissue homogenization in combination with a bland trypsin treatment and Percoll gradient purification to isolate single neuronal mitochondria from the spinal cord of a transgenic mouse in which a cyan fluorescent protein (CFP) was fused to a mitochondrial transport sequence derived from cytochrome c and expressed in neurons unde...

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