CRISPR knockdown of Kcnq3 attenuates the M-current and increases excitability of NPY/AgRP neurons to alter energy balance

Mol Metab. 2021 Jul:49:101218. doi: 10.1016/j.molmet.2021.101218. Epub 2021 Mar 22.

Abstract

Objective: Arcuate nucleus neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior. The M-current, a subthreshold non-inactivating potassium current, plays a critical role in regulating NPY/AgRP neuronal excitability. Fasting decreases while 17β-estradiol increases the M-current by regulating the mRNA expression of Kcnq2, 3, and 5 (Kv7.2, 3, and 5) channel subunits. Incorporating KCNQ3 into heteromeric channels has been considered essential to generate a robust M-current. Therefore, we investigated the behavioral and physiological effects of selective Kcnq3 deletion from NPY/AgRP neurons.

Methods: We used a single adeno-associated viral vector containing a recombinase-dependent Staphylococcus aureus Cas9 with a single-guide RNA to selectively delete Kcnq3 in NPY/AgRP neurons. Single-cell quantitative measurements of mRNA expression and whole-cell patch clamp experiments were conducted to validate the selective knockdown. Body weight, food intake, and locomotor activity were measured in male mice to assess disruptions in energy balance.

Results: The virus reduced the expression of Kcnq3 mRNA without affecting Kcnq2 or Kcnq5. The M-current was attenuated, causing NPY/AgRP neurons to be more depolarized, exhibit a higher input resistance, and require less depolarizing current to fire action potentials, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it significantly reduced the locomotor activity as measured by open-field testing. Control mice on a high-fat diet exhibited an enhanced M-current and increased Kcnq2 and Kcnq3 expression, but the M-current remained significantly attenuated in KCNQ3 knockdown animals.

Conclusions: The M-current plays a critical role in modulating the intrinsic excitability of NPY/AgRP neurons that is essential for maintaining energy homeostasis.

Keywords: 17β-estradiol; Agouti-related peptide; Energy balance; High-fat diet; KCNQ channels; Neuropeptide Y.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Action Potentials
  • Agouti-Related Protein / metabolism*
  • Animals
  • Arcuate Nucleus of Hypothalamus / metabolism*
  • Body Weight
  • CRISPR-Cas Systems
  • Diet, High-Fat
  • Energy Metabolism / physiology*
  • Estradiol / metabolism
  • Fasting
  • Feeding Behavior
  • Female
  • KCNQ3 Potassium Channel / genetics*
  • KCNQ3 Potassium Channel / metabolism*
  • Male
  • Mice
  • Neurons / metabolism*
  • Neuropeptide Y / genetics
  • Neuropeptide Y / metabolism*
  • Neuropeptides / metabolism*

Substances

  • Agouti-Related Protein
  • KCNQ3 Potassium Channel
  • Kcnq3 protein, mouse
  • Neuropeptide Y
  • Neuropeptides
  • Estradiol