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. 2009 Feb 25;29(8):2528-33.
doi: 10.1523/JNEUROSCI.5764-08.2009.

Glucose inhibition persists in hypothalamic neurons lacking tandem-pore K+ channels

Alice Guyon  1 , Magalie P Tardy, Carole Rovère, Jean-Louis Nahon, Jacques Barhanin, Florian Lesage
Affiliations

Glucose inhibition persists in hypothalamic neurons lacking tandem-pore K+ channels

Alice Guyon et al. J Neurosci. .

Abstract

Glucose sensing by hypothalamic neurons triggers adaptive metabolic and behavioral responses. In orexin neurons, extracellular glucose activates a leak K(+) current promoting electrical activity inhibition. Sensitivity to external acidification and halothane, and resistance to ruthenium red designated the tandem-pore K(+) (K(2P)) channel subunit TASK3 as part of the glucose-induced channel. Here, we show that glucose inhibition and its pH sensitivity persist in mice lacking TASK3 or TASK1, or both subunits. We also tested the implication of another class of K(2P) channels activated by halothane. In the corresponding TREK1/2/TRAAK triple knock-out mice, glucose inhibition persisted in hypothalamic neurons ruling out a major contribution of these subunits to the glucose-activated K(+) conductance. Finally, block of this glucose-induced hyperpolarizing current by low Ba(2+) concentrations was consistent with the conclusion that K(2P) channels are not required for glucosensing in hypothalamic neurons.

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Figures

Figure 1.
Figure 1.
TASK3 gene inactivation. A, Inactivation strategy. Open triangles represent loxP sequences. Exon 2 is deleted in KO mice. B, PCR analysis of genomic DNA. Primer positions are shown in A. C, TASK3 immunodetection in adult mouse cerebellum. TASK3 expression observed in the molecular layer (Mol) of wild-type (WT) mouse was lacking in KO mouse. Gr, Granule cell layer; Pk, Purkinje cells.
Figure 2.
Figure 2.
Responses to glucose of orexin neurons in K2P channel KO mice. A, Typical current response of a orexin neuron recorded in the voltage-clamp mode in response to a 500 ms hyperpolarizing pulse to −100 mV from a holding potential of −60 mV. Notice the presence of a slowly activating inward current (Ih). B, Superimposed typical responses to depolarizing and hyperpolarizing pulses delivered from the resting potential of a orexin neuron recorded in the current-clamp mode. Notice that the discharge of action potential shows very few adaptation. C, Examples of typical pattern obtained in single cell multiplex RT-PCR from one presumed orexin neuron. Multiplex RT-PCR was performed as described by Guyon et al. (2005). A negative control without reverse transcriptase (NRT) was performed. D, Current-clamp recordings showing the effect of increases in glucose concentration from 0.2 to 4.5 mm in double TASK1−/−TASK3−/− (TASK−/−) and triple TREK1−/−TREK2−/−TRAAK−/− (TREK−/−) orexin neurons. E, Mean maximum amplitude (+SEM) of the hyperpolarization recorded in control and KO mice (number of neurons tested in parenthesis).
Figure 3.
Figure 3.
Pharmacology of the glucose-response in orexin neurons. A, B, Current-clamp recordings showing the effect of external pH (pHe) on glucose-induced hyperpolarization in WT (A) and TASK−/− (B) neurons. C, D, Responses to glucose are reversed by Ba2+. Examples of the effects of 200 μm Ba2+ (C) or increasing Ba2+ concentrations (D) are shown. Recordings were obtained in control C57BL/6J mice.
Figure 4.
Figure 4.
TREK2 gene inactivation. A, Inactivation strategy. Exon 2 is duplicated in KO mice introducing a frameshift resulting in a premature stop codon. B, PCR analysis of genomic DNA from mutant mice. Primer positions are shown in A. C, RT-PCR analysis of brain cDNAs. Primer positions are shown in A. Multiple PCR products are amplified from KO animals that correspond to alternative hybridizations from the exon 2 repeat. D, Western analysis of adult mouse brain proteins using anti-TREK2 antibodies.
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