BK channels sustain neuronal Ca2+ oscillations to support hippocampal long-term potentiation and memory formation.

Mutations of large conductance Ca2+- and voltage-activated K+ channels (BK) are associated with cognitive impairment. Here we report that CA1 pyramidal neuron-specific conditional BK knock-out (cKO) mice display normal locomotor and anxiety behavior. They do, however, exhibit impaired memory acquisition and retrieval in the Morris Water Maze (MWM) when compared to littermate controls (CTRL). In line with cognitive impairment in vivo, electrical and chemical long-term potentiation (LTP) in cKO brain slices were impaired in vitro. We further used a genetically encoded fluorescent K+ biosensor and a Ca2+-sensitive probe to observe cultured hippocampal neurons during chemical LTP (cLTP) induction. cLTP massively reduced intracellular K+ concentration ([K+]i) while elevating L-Type Ca2+ channel- and NMDA receptor-dependent Ca2+ oscillation frequencies. Both, [K+]i decrease and Ca2+ oscillation frequency increase were absent after pharmacological BK inhibition or in cells lacking BK. Our data suggest that L-Type- and NMDAR-dependent BK-mediated K+ outflow significantly contributes to hippocampal LTP, as well as learning and memory.

Slack K+ channels attenuate NMDA-induced excitotoxic brain damage and neuronal cell death.

The neuronal Na+ -activated K+ channel Slack (aka Slo2.2, KNa 1.1, or Kcnt1) has been implicated in setting and maintaining the resting membrane potential and defining excitability and firing patterns, as well as in the generation of the slow afterhyperpolarization following bursts of action potentials. Slack activity increases significantly under conditions of high intracellular Na+ levels, suggesting this channel may exert important pathophysiological functions. To address these putative roles, we studied whether Slack K+ channels contribute to pathological changes and excitotoxic cell death caused by glutamatergic overstimulation of Ca2+ - and Na+ -permeable N-methyl-D-aspartic acid receptors (NMDAR). Slack-deficient (Slack KO) and wild-type (WT) mice were subjected to intrastriatal microinjections of the NMDAR agonist NMDA. NMDA-induced brain lesions were significantly increased in Slack KO vs WT mice, suggesting that the lack of Slack renders neurons particularly susceptible to excitotoxicity. Accordingly, excessive neuronal cell death was seen in Slack-deficient primary cerebellar granule cell (CGC) cultures exposed to glutamate and NMDA. Differences in neuronal survival between WT and Slack KO CGCs were largely abolished by the NMDAR antagonist MK-801, but not by NBQX, a potent and highly selective competitive antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, NMDAR-evoked Ca2+ signals did not differ with regard to Slack genotype in CGCs. However, real-time monitoring of K+ following NMDAR activation revealed a significant contribution of this channel to the intracellular drop in K+ . Finally, TrkB and TrkC neurotrophin receptor transcript levels were elevated in NMDA-exposed Slack-proficient CGCs, suggesting a mechanism by which this K+ channel contributes to the activation of the extracellular-signal-regulated kinase (Erk) pathway and thereby to neuroprotection. Combined, our findings suggest that Slack-dependent K+ signals oppose the NMDAR-mediated excitotoxic neuronal injury by promoting pro-survival signaling via the BDNF/TrkB and Erk axis.

The Na+-activated K+ channel Slack contributes to synaptic development and plasticity.

Human mutations of the Na+-activated K+ channel Slack (KCNT1) are associated with epilepsy and intellectual disability. Accordingly, Slack knockout mice (Slack-/-) exhibit cognitive flexibility deficits in distinct behavioral tasks. So far, however, the underlying causes as well as the role of Slack in hippocampus-dependent memory functions remain enigmatic. We now report that infant (P6-P14) Slack-/- lack both hippocampal LTD and LTP, likely due to impaired NMDA receptor (NMDAR) signaling. Postsynaptic GluN2B levels are reduced in infant Slack-/-, evidenced by lower amplitudes of NMDAR-meditated excitatory postsynaptic potentials. Low GluN2B affected NMDAR-mediated Ca2+-influx, rendering cultured hippocampal Slack-/-neurons highly insensitive to the GluN2B-specific inhibitor Ro 25-6981. Furthermore, dephosphorylation of the AMPA receptor (AMPAR) subunit GluA1 at S845, which is involved in AMPAR endocytosis during homeostatic and neuromodulator-regulated plasticity, is reduced after chemical LTD (cLTD) in infant Slack-/-. We additionally detect a lack of mGluR-induced LTD in infant Slack-/-, possibly caused by upregulation of the recycling endosome-associated small GTPase Rab4 which might accelerate AMPAR recycling from early endosomes. Interestingly, LTP and mGluR LTD, but not LTD and S845 dephosphorylation after cLTD are restored in adult Slack-/-. This together with normalized expression levels of GluN2B and Rab4 hints to developmental "restoration" of LTP expression despite Slack ablation, whereas in infant and adult brain, NMDAR-dependent LTD induction depends on this channel. Based on the present findings, NMDAR and vesicular transport might represent novel targets for the therapy of intellectual disability associated with Slack mutations. Consequently, careful modulation of hippocampal Slack activity should also improve learning abilities.

Functional Coupling of Slack Channels and P2X3 Receptors Contributes to Neuropathic Pain Processing.

The sodium-activated potassium channel Slack (KNa1.1, Slo2.2, or Kcnt1) is highly expressed in populations of sensory neurons, where it mediates the sodium-activated potassium current (IKNa) and modulates neuronal activity. Previous studies suggest that Slack is involved in the processing of neuropathic pain. However, mechanisms underlying the regulation of Slack activity in this context are poorly understood. Using whole-cell patch-clamp recordings we found that Slack-mediated IKNa in sensory neurons of mice is reduced after peripheral nerve injury, thereby contributing to neuropathic pain hypersensitivity. Interestingly, Slack is closely associated with ATP-sensitive P2X3 receptors in a population of sensory neurons. In vitro experiments revealed that Slack-mediated IKNa may be bidirectionally modulated in response to P2X3 activation. Moreover, mice lacking Slack show altered nocifensive responses to P2X3 stimulation. Our study identifies P2X3/Slack signaling as a mechanism contributing to hypersensitivity after peripheral nerve injury and proposes a potential novel strategy for treatment of neuropathic pain.

Slack K+ channels confer protection against myocardial ischemia/reperfusion injury.

AIMS: Na+-activated Slack potassium (K+) channels are increasingly recognized as regulators of neuronal activity, yet little is known about their role in the cardiovascular system. Slack activity increases when intracellular Na+ concentration ([Na+]i) reaches pathophysiological levels. Elevated [Na+]i is a major determinant of the ischemia and reperfusion (I/R)-induced myocardial injury, thus we hypothesized that Slack plays a role under these conditions. METHODS: and results: K+ currents in cardiomyocytes (CMs) obtained from wildtype (WT) but not from global Slack knockout (KO) mice were sensitive to electrical inactivation of voltage-sensitive Na+-channels. Live-cell imaging demonstrated that K+ fluxes across the sarcolemma rely on Slack, while the depolarized resting membrane potential in Slack-deficient CMs led to excessive cytosolic Ca2+ accumulation and finally to hypoxia/reoxygenation-induced cell death. Cardiac damage in an in vivo model of I/R was exacerbated in global and CM-specific conditional Slack mutants and largely insensitive to mechanical conditioning maneuvers. Finally, the protection conferred by mitochondrial ATP-dependent K+ channels required functional Slack in CMs. CONCLUSIONS: Collectively, our study provides evidence for Slack's crucial involvement in the ion homeostasis of no or low O2-stressed CMs. Thereby, Slack activity opposes the I/R-induced fatal Ca2+-uptake to CMs supporting the cardioprotective signaling widely attributed to mitoKATP function.

Slack K+ channels limit kainic acid-induced seizure severity in mice by modulating neuronal excitability and firing.

Mutations of the Na+-activated K+ channel Slack (KCNT1) are associated with terrible epilepsy syndromes that already begin in infancy. Here we report increased severity of acute kainic acid-induced seizures in adult and juvenile Slack knockout mice (Slack-/-) in vivo. Fittingly, we find exacerbation of cell death following kainic acid exposure in organotypic hippocampal slices as well as dissociated hippocampal cultures from Slack-/- in vitro. Furthermore, in cultured Slack-/- neurons, kainic acid-triggered Ca2+ influx and K+ efflux as well as depolarization-induced tetrodotoxin-sensitive inward currents are higher compared to the respective controls. This apparent changes in ion homeostasis could possibly explain altered action potential kinetics of Slack-/- neurons: steeper rise slope, decreased threshold, and duration of afterhyperpolarization, which ultimately lead to higher action potential frequencies during kainic acid application or injection of depolarizing currents. Based on our data, we propose Slack as crucial gatekeeper of neuronal excitability to acutely limit seizure severity.

Loss of Sodium-Activated Potassium Channel Slack and FMRP Differentially Affect Social Behavior in Mice.

The sodium-activated potassium channel Slack (Slo2.2) is widely expressed in central and peripheral neurons where it is supposed to shape firing properties important for neuronal excitability. Slack activity is enhanced by interaction with the Fragile-X-Mental-Retardation-Protein (FMRP) and loss of FMRP leads to decreased sodium-activated potassium currents in medial nucleus of the trapezoid body neurons of the Fmr1-knockout (KO) mouse representing a mouse model of the human Fragile-X-Syndrome (FXS) and autism. Autism is a frequent comorbidity of FXS, but it is unclear whether Slack is involved in autistic or related conditions of FXS in vivo. By applying a wide range of behavioral tests, we compared social and autism-related behaviors in Slack- and FMRP-deficient mice. In our hands, as expected, FMRP-deficiency causes autism-related behavioral changes in nesting and in a marble-burying test. In contrast, Slack-deficient males exhibited specific abnormalities in sociability in direct and indirect social interaction tests. Hence, we show for the first time that a proper Slack channel function is mandatory for normal social behavior in mice. Nevertheless, as deficits in social behaviors seem to occur independently from each other in FMRP and Slack null mutants, we conclude that Slack is not involved in the autistic phenotype of FMRP KO mice.