Mitochondrial Ca2+Uptake Regulates the Excitability of Myenteric Neurons

Abstract

We investigated the role of mitochondria in the regulation of intracellular Ca²⁺([Ca²⁺]_i) and excitability of myenteric neurons in guinea pig ileum, using microelectrodes and fura-2 [Ca²⁺]_i measurements. In AH/Type-II neurons, action potentials evoke ryanodine-sensitive increases in [Ca²⁺]_i that activate Ca²⁺-dependent K⁺ channels and slow afterhyperpolarizations (AH) lasting ∼15 sec. Exposure to the protonophore carbonyl cyanide<i>p</i>-(trifluoromethoxy)phenylhydrazone (FCCP; 1 μm) had no significant effect on the membrane potential or resting [Ca²⁺]_i. However, action potentials elicited in the presence of FCCP triggered a sustained (&gt;5 min) increase in [Ca²⁺]_i and a compound hyperpolarization (13.4 ± 1.5 mV). The respiratory chain blockers antimycin A and rotenone (10 μm) had similar effects that developed more slowly. Depletion of the intracellular Ca²⁺ stores with thapsigargin (2 μm) or ryanodine (10 μm) greatly attenuated the hyperpolarization caused by FCCP. S/Type-I neurons that do not have AH were hyperpolarized by mitochondrial inhibition independently of action potentials. Blockade of the F₀F₁ ATPase by oligomycin (10 μm) had variable effects on myenteric neurons. The majority of AH/Type-II neurons were hyperpolarized by oligomycin, most likely by activating ATP-dependent K⁺ channels. This hyperpolarization was not triggered by action potential firing and not accompanied by an increase in [Ca²⁺]_i. MitoTracker staining revealed a dense mitochondrial network particularly in myenteric AH/Type-II neurons, supporting the importance of mitochondrial Ca²⁺ buffering in this subset of neurons. The data indicate that mitochondrial uptake of Ca²⁺released from the endoplasmic reticulum sets [Ca²⁺]_i and the activity of Ca²⁺-dependent conductances, thus regulating the excitability of myenteric neurons.