Potassium Leak Channels and Mitochondrial Complex I Interact in Glutamatergic Interneurons of the Mouse Spinal Cord

Abstract

Background: Volatile anesthetics induce hyperpolarizing potassium currents in spinal cord neurons that may contribute to their mechanism of action. They are induced at lower concentrations of isoflurane in noncholinergic neurons from mice carrying a loss-of-function mutation of the Ndufs4 gene, required for mitochondrial complex I function. The yeast NADH dehydrogenase enzyme, NDi1, can restore mitochondrial function in the absence of normal complex I activity, and gain-of-function Ndi1 transgenic mice are resistant to volatile anesthetics. The authors tested whether NDi1 would reduce the hyperpolarization caused by isoflurane in neurons from Ndufs4 and wild-type mice. Since volatile anesthetic behavioral hypersensitivity in Ndufs4 is transduced uniquely by glutamatergic neurons, it was also tested whether these currents were also unique to glutamatergic neurons in the Ndufs4 spinal cord.

Methods: Spinal cord neurons from wild-type, NDi1, and Ndufs4 mice were patch clamped to characterize isoflurane sensitive currents. Neuron types were marked using fluorescent markers for cholinergic, glutamatergic, and γ-aminobutyric acid-mediated (GABAergic) neurons. Norfluoxetine was used to identify potassium channel type. Neuron type-specific Ndufs4 knockout animals were generated using type-specific Cre-recombinase with floxed Ndufs4.

Results: Resting membrane potentials (RMPs) of neurons from NDi1;Ndufs4, unlike those from Ndufs4, were not hyperpolarized by 0.6% isoflurane (Ndufs4, ΔRMP -8.2 mV [-10 to -6.6]; P = 1.3e-07; Ndi1;Ndufs4, ΔRMP -2.1 mV [-7.6 to +1.4]; P = 1). Neurons from NDi1 animals in a wild-type background were not hyperpolarized by 1.8% isoflurane (wild-type, ΔRMP, -5.2 mV [-7.3 to -3.2]; P = 0.00057; Ndi1, ΔRMP, 0.6 mV [-1.7 to 3.2]; P = 0.68). In spinal cord slices from global Ndufs4 animals, holding currents (HC) were induced by 0.6% isoflurane in both GABAergic (ΔHC, 81.3 pA [61.7 to 101.4]; P = 2.6e-05) and glutamatergic (ΔHC, 101.2 pA [63.0 to 146.2]; P = 0.0076) neurons. In neuron type-specific Ndufs4 knockouts, HCs were increased in cholinergic (ΔHC, 119.5 pA [82.3 to 156.7]; P = 0.00019) and trended toward increase in glutamatergic (ΔHC, 85.5 pA [49 to 126.9]; P = 0.064) neurons but not in GABAergic neurons.

Conclusions: Bypassing complex I by overexpression of NDi1 eliminates increases in potassium currents induced by isoflurane in the spinal cord. The isoflurane-induced potassium currents in glutamatergic neurons represent a potential downstream mechanism of complex I inhibition in determining minimum alveolar concentration.