Knockout of Slo2.2 enhances itch, abolishes KNa current, and increases action potential firing frequency in DRG neurons

Abstract

Two mammalian genes, Kcnt1 and Kcnt2, encode pore-forming subunits of Na+-dependent K+ (KNa) channels. Progress in understanding KNa channels has been hampered by the absence of specific tools and methods for rigorous KNa identification in native cells. Here, we report the genetic disruption of both Kcnt1 and Kcnt2, confirm the loss of Slo2.2 and Slo2.1 protein, respectively, in KO animals, and define tissues enriched in Slo2 expression. Noting the prevalence of Slo2.2 in dorsal root ganglion, we find that KO of Slo2.2, but not Slo2.1, results in enhanced itch and pain responses. In dissociated small diameter DRG neurons, KO of Slo2.2, but not Slo2.1, abolishes KNa current. Utilizing isolectin B4+ neurons, the absence of KNa current results in an increase in action potential (AP) firing and a decrease in AP threshold. Activation of KNa acts as a brake to initiation of the first depolarization-elicited AP with no discernible effect on afterhyperpolarizations.DOI: http://dx.doi.org/10.7554/eLife.10013.001

Cardiac Slo2.1 Is Required for Volatile Anesthetic Stimulation of K+ Transport and Anesthetic Preconditioning

“…22 However, in mammals Slo2 has diverged into two paralogs ( Slo2.1 and Slo2.2 ) with differing ion sensitivity to the C. elegans channel. Using a recently developed mouse strain with the genes coding for Slick ( Kcnt2 or Slo2.1 ) and Slack ( Kcnt1 or Slo2.2 ) both deleted, 28 our goal herein was to determine if either or both of these mammalian gene products are orthologous to the worm Slo2 channel, and facilitate K + flux across the mitochondrial inner membrane in response to VA. First, we verified using genomic PCR that the Slo2.x double knockout (dKO) contained the expected lesions in each of the two genes, and that each allele was unambiguously detectable (Figures 2A and 2B). Next, we used Western blot analysis to query whether the channels themselves were similarly ablated.…”

“…However, it is likely that bithionol stimulates other channel activities or even mitochondrial function through unknown mechanisms of action, and very low levels of Slo2.2 are expressed in the heart. 28 …”

“…All procedures were approved by the University of Rochester’s Committee on Animal Resources (protocol #2010-030) in accordance with the NIH Guide for the Care and Use of Laboratory Animals (2011 revision). A Slo2.x dKO strain containing both mutant Slo2.x alleles 28 was provided by Dr. Chris Lingle, Ph.D (Washington University, St. Louis MO). Mice were out-crossed >6x to a C57/BL6 background, and conventional breeding was employed such that experimental WT and knockout mice (males, 8–10 weeks old) were littermates.…”

The Phe932Ile mutation in KCNT1 channels associated with severe epilepsy, delayed myelination and leukoencephalopathy produces a loss-of-function channel phenotype

“…In addition, the Slack monoclonal antibody used in this study has been validated by multiple labs for biochemical analyses. (Bansal & Fisher, 2016; Gururaj et al, 2016; Lu et al, 2013; Martinez-Espinosa et al, 2015; Rizzi et al, 2016). This antibody only weakly reacts with the human channel due to amino acid differences in the epitope, necessitating the use of the rat variant.…”

International Union of Basic and Clinical Pharmacology. C. Nomenclature and Properties of Calcium-Activated and Sodium-Activated Potassium Channels

“…The channel is also expressed at high levels in nociceptive neurons within the dorsal root ganglion, and suppression of current using several approaches, including deletion of the gene for K Na 1.1, enhances neuronal excitability and produces hypersensitivity of animals to several pain-inducing stimuli (Gao et al, 2008;Tamsett et al, 2009;Nuwer et al, 2010;Huang et al, 2013;Lu et al, 2015;Martinez-Espinosa et al, 2015). Deletion of the gene in mice also has a variety of more wide-ranging effects on behavior, including an inability to reverse a previously learned behavior (Bausch et al, 2015).…”