Mitochondrial Ca2+ Dynamics Reveals Limited Intramitochondrial Ca2+ Diffusion

¹ Semmelweis University, Dept. Medical Biochemistry, Neurobiochemical Group

^* To whom correspondence should be addressed. E-mail: av{at}puskin.sote.hu.

Submitted on August 2, 2004
Revised on September 7, 2004
Accepted on 13 October 2004

	Abstract

To reveal heterogeneity of mitochondrial function on single mitochondrion level we have studied the spatiotemporal dynamics of the mitochondrial Ca2+ signaling and the mitochondrial membrane potential using wide-field fluorescence imaging and digital image processing techniques. Here we demonstrate first time discrete sites - intramitochondrial hotspots - of Ca2+ uptake following Ca2+ release from intracellular stores, and spreading of Ca2+ rise within the mitochondria. The phenomenon was characterized by comparison of observations in intact cells stimulated by ATP and in plasma membrane permeabilized or in ionophore-treated cells exposed to elevated buffer [Ca2+]. The findings indicate that Ca2+ diffuses laterally within the mitochondria, and that the diffusion is limited for shorter segments of the mitochondrial network. These observations were supported by mathematical simulation of buffered diffusion. The mitochondrial membrane potential was investigated using the potentiometric dye TMRM. Irradiation-induced fluctuations (flickering) of TMRM fluorescence showed synchronicity over large regions of the mitochondrial network, indicating that certain parts of this network form electrical syncytia. The spatial extension of these syncytia was decreased by 2-aminoethoxydiphenyl borate (2-APB) or by propranolol (blockers of non-classical mitochondrial permeabilities). Our data suggest that mitochondria form syncytia of electrical conductance while the passage of Ca2+ is restricted to the individual organelle.

Key Words: Calcium Signaling, Computer-Assisted Image Processing, Mitochondrial Membrane Potential, Modeling, Permeability Transition Pore, Rhod-2

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