Increased large conductance calcium-activated potassium (BK) channel expression accompanied by STREX variant downregulation in the developing mouse CNS

BACKGROUND: Large conductance calcium- and voltage activated potassium (BK) channels are important determinants of neuronal excitability through effects on action potential duration, frequency and synaptic efficacy. The pore- forming subunits are encoded by a single gene, KCNMA1, which undergoes extensive alternative pre mRNA splicing. Different splice variants can confer distinct properties on BK channels. For example, insertion of the 58 amino acid stress-regulated exon (STREX) insert, that is conserved throughout vertebrate evolution, encodes channels with distinct calcium sensitivity and regulation by diverse signalling pathways compared to the insertless (ZERO) variant. Thus, expression of distinct splice variants may allow cells to differentially shape their electrical properties during development. However, whether differential splicing of BK channel variants occurs during development of the mammalian CNS has not been examined. RESULTS: Using quantitative real-time polymerase chain reaction (RT-PCR) Taqman™ assays, we demonstrate that total BK channel transcripts are up regulated throughout the murine CNS during embryonic and postnatal development with regional variation in transcript levels. This upregulation is associated with a decrease in STREX variant mRNA expression and an upregulation in ZERO variant expression. CONCLUSION: As BK channel splice variants encode channels with distinct functional properties the switch in splicing from the STREX phenotype to ZERO phenotype during embryonic and postnatal CNS development may provide a mechanism to allow BK channels to control distinct functions at different times of mammalian brain development

Palmitoylation of the β4-Subunit Regulates Surface Expression of Large Conductance Calcium-activated Potassium Channel Splice Variants

Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels

Control of hypothalamic-pituitary-adrenal stress axis activity by the intermediate conductance calcium-activated potassium channel, SK4

NON-TECHNICAL SUMMARY: Our ability to respond to stress is critically dependent upon the release of the stress hormone adrenocorticotrophic hormone (ACTH) from corticotroph cells of the anterior pituitary gland. ACTH release is controlled by the electrical properties of corticotrophs that are determined by the movement of ions through channel pores in the plasma membrane. We show that a calcium-activated potassium ion channel called SK4 is expressed in corticotrophs and regulates ACTH release. We provide evidence of how SK4 channels control corticotroph function, which is essential for understanding homeostasis and for treating stress-related disorders. ABSTRACT: The anterior pituitary corticotroph is a major control point for the regulation of the hypothalamic–pituitary–adrenal (HPA) axis and the neuroendocrine response to stress. Although corticotrophs are known to be electrically excitable, ion channels controlling the electrical properties of corticotrophs are poorly understood. Here, we exploited a lentiviral transduction system to allow the unequivocal identification of live murine corticotrophs in culture. We demonstrate that corticotrophs display highly heterogeneous spontaneous action-potential firing patterns and their resting membrane potential is modulated by a background sodium conductance. Physiological concentrations of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) cause a depolarization of corticotrophs, leading to a sustained increase in action potential firing. A major component of the outward potassium conductance was mediated via intermediate conductance calcium-activated (SK4) potassium channels. Inhibition of SK4 channels with TRAM-34 resulted in an increase in corticotroph excitability and exaggerated CRH/AVP-stimulated ACTH secretion in vitro. In accordance with a physiological role for SK4 channels in vivo, restraint stress-induced plasma ACTH and corticosterone concentrations were significantly enhanced in gene-targeted mice lacking SK4 channels (Kcnn4(−/−)). In addition, Kcnn4(−/−) mutant mice displayed enhanced hypothalamic c-fos and nur77 mRNA expression following restraint, suggesting increased neuronal activation. Thus, stress hyperresponsiveness observed in Kcnn4(−/−) mice results from enhanced secretagogue-induced ACTH output from anterior pituitary corticotrophs and may also involve increased hypothalamic drive, thereby suggesting an important role for SK4 channels in HPA axis function

Palabras de apertura del seminario de investigación del Instituto Iberoamericano

Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) induces neuroprotection against excitotoxic damage in primary cortical neurons via sustained nuclear factor-kappa B (NF-kappa B) activation. The transcription factor NF-kappa B can regulate the expression of small conductance calcium-activated potassium (K(Ca)) channels. These channels reduce neuronal excitability and as such may yield neuroprotection against neuronal overstimulation. In the present study we investigated whether TNF-alpha-mediated neuroprotective signaling is inducing changes in the expression of small conductance K(Ca) channels. Interestingly, the expression of K(Ca)2.2 channel was up-regulated by TNF-alpha treatment in a time-dependent manner whereas the expression of K(Ca)2.1 and K(Ca)2.3 channels was not altered. The increase in K(Ca)2.2 channel expression after TNF-alpha treatment was shown to be dependent on TNF-R2 and NF-kappa B activation. Furthermore, activation of small conductance K(Ca) channels by 6,7-dichloro-1H-indole-2,3-dione 3-oxime or cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine-induced neuroprotection against a glutamate challenge. Treatment with the small conductance K(Ca) channel blocker apamin or K(Ca)2.2 channel siRNA reverted the neuroprotective effect elicited by TNF-alpha. We conclude that treatment of primary cortical neurons with TNF-alpha leads to increased K(Ca)2.2 channel expression which renders neurons more resistant to excitotoxic cell death

Small-conductance Ca2+-activated potassium type 2 channels regulate the formation of contextual fear memory.

Small-conductance, Ca2+ activated K+ channels (SK channels) are expressed at high levels in brain regions responsible for learning and memory. In the current study we characterized the contribution of SK2 channels to synaptic plasticity and to different phases of hippocampal memory formation. Selective SK2 antisense-treatment facilitated basal synaptic transmission and theta-burst induced LTP in hippocampal brain slices. Using the selective SK2 antagonist Lei-Dab7 or SK2 antisense probes, we found that hippocampal SK2 channels are critical during two different time windows: 1) blockade of SK2 channels before the training impaired fear memory, whereas, 2) blockade of SK2 channels immediately after the training enhanced contextual fear memory. We provided the evidence that the post-training cleavage of the SK2 channels was responsible for the observed bidirectional effect of SK2 channel blockade on memory consolidation. Thus, Lei-Dab7-injection before training impaired the C-terminal cleavage of SK2 channels, while Lei-Dab7 given immediately after training facilitated the C-terminal cleavage. Application of the synthetic peptide comprising a leucine-zipper domain of the C-terminal fragment to Jurkat cells impaired SK2 channel-mediated currents, indicating that the endogenously cleaved fragment might exert its effects on memory formation by blocking SK2 channel-mediated currents. Our present findings suggest that SK2 channel proteins contribute to synaptic plasticity and memory not only as ion channels but also by additionally generating a SK2 C-terminal fragment, involved in both processes. The modulation of fear memory by down-regulating SK2 C-terminal cleavage might have applicability in the treatment of anxiety disorders in which fear conditioning is enhanced

Cleavage of SK2 channel protein after contextual fear conditioning training.

<p>Western blot analysis of SK2 protein levels in (A) hippocampal tissue obtained from naïve mice or 1 h and 3 h after training (context+shock). In the context group, mice were exposed to the training context without receiving a foot-shock. In the shock group, mice received a foot-shock immediately after they were exposed to the training context and were removed immediately after the foot-shock. Experiment was performed twice. (B) Representative immunoblot of SK2 protein levels in hippocampal tissue from naïve, trained and trained vehicle- or Lei-Dab⁷-injected mice. Mice were injected either 0.5 h before or 0 h after training as indicated. Hippocampal tissue was removed 1 h after training. The SK2_(538–555) antibody recognized the 64-kDa SK2 protein and a 10-kDa SK2 C-terminal fragment (top). (C) SK2 protein levels in hippocampal tissue from naïve non-injected, Lei-Dab⁷-injected naïve, or vehicle-injected trained mice. Hippocampal tissue was removed 90 or 210 min after injection (1h and/or 3h after training). The number of individual samples per treatment was five. Data presented are the mean ± SEM. Statistically significant differences: *<i>p</i> < 0.05 versus naive, ^#<i>p</i> < 0.005 versus 3 hours context and conditioning groups, ^a<i>p</i><0.05 versus training + vehicle.</p

SK2 leucine zipper (SK2-LZ) domain peptide enhances fear conditioning and TBS-LTP by inhibiting SK2 current.

<p>(A) Mice intrahippocampally (i.h.) injected at 30 before or immediately after training with either the SK2-LZ peptide _(488–526), a random sequenced control peptide or vehicle showed no difference in freezing when compared to non-injected animals. Injection of the selective SK2 antagonist Lei-Dab⁷ 0.5 h before training impaired freezing when compared to non-injected animals. This impairment was rescued if mice were injected with the SK2-LZ peptide _(488–526) immediately after training. I.h. injection of SK2-LZ peptide _(488–526) alone, either 30 min before training or immediately following training did not affect contextual fear. Freezing was measured in the memory test 24 h after training. n = 7–10 mice/group. Statistics were performed by one-way ANOVA with Bonferroni multiple comparisons test (*<i>p</i> < 0.05). (B) TBS-LTP elicited in slices from mice that were pre-treated with SK2-LZ peptide _(488–526) was significantly enhanced when compared to LTP induced in slices from naive mice. There was no statistical difference between control peptide-injected mice and naïve mice. Statistics were performed by two-way ANOVA with Bonferroni multiple comparisons test (*<i>p</i> < 0.05). Insets: Responses shown are fEPSPs recorded during baseline (upper row) and 55–60 min (bottom row) after the induction of LTP (post-induction). Traces are averages of five consecutive responses. (C) Average whole-cell currents were recorded in Jurkat cells endogenously expressing SK2 channels. Voltage ramps were from -100 mV to 100 mV for 100 ms delivered at 2 s intervals. SK2 current was measured at 80 mV and normalized to cell size (in pF) to show current density over time. After 200 seconds external solutions containing 100 μM SK2-LZ peptide (filled circles, n = 6) was applied to the cells for 150 seconds or no external solution application (control; open circles, n = 4). Arrows at 196 s and 348 s indicate the time points at which the current-voltage (I-V) curves in D were obtained (D) Representative I-V curves for endogenous SK2 in Jurkat cells taken after 196 s, right before application (dotted line) and at the end of the experiment at 348 s (solid line) in unexposed control cells (left panel) or when exposed to 100 μM SK2-LZ peptide. Data presented are the mean ± SEM.</p

Enhanced basal synaptic transmission and LTP in hippocampal slices from mice pre-treated with SK2 antisense ODNs.

<p>The distance between the stimulating and recording electrodes was kept constant between slices. (A) Input-output curve of fEPSP slope (mV/ms) versus stimulus (V) at the SC-CA1 pyramidal cell synapse in naive mice and mice pre-treated with vehicle, antisense ODNs against SK2 channels and control ODNs. The maximal fEPSP slopes were significantly larger in the SK2 antisense-treated mice than those in the naive, vehicle and control ODNs-treated mice. Data are presented as mean ± s.e.m. (B) Relationship between the slope of the evoked fEPSPs from panel A and the corresponding fiber volley amplitude. SK2 antisense-treated mice exhibit a greater postsynaptic response than control groups to similar presynaptic depolarization. Data are presented as mean ± s.e.m. (C) Comparison of PPF in naive mice and mice pre-treated with vehicle, antisense ODNs against SK2 channels and control ODNs. No differences were found between these four groups of mice. Data presented are the mean ± SEM of the facilitation of the second response relative to the first response. Insets: Field EPSPs recorded in response to paired-pulse stimulation at an interstimulus interval of 50 ms in slices from all four treatment groups as indicated. (D) TBS-LTP elicited in slices from mice that were pre-treated with SK2 antisense ODNs was significantly enhanced when compared to LTP induced in slices from naive mice. There was no statistical difference between control ODNs-injected mice and mice that were pre-injected with vehicle. Insets: Responses shown are fEPSPs recorded during baseline (upper row) and 55–60 min (bottom row) after the induction of LTP (post-induction). Traces are averages of five consecutive responses. Statistical significance was determined by two-way ANOVA followed by Bonferroni multiple comparisons test (*<i>p</i> < 0.05).</p

Pre-exposure of mice overcomes impaired contextual fear after inhibition of SK2 channel function.

<p>Foot-shock intensity was reduced from 0.7 mA to 0.5 mA. (A) Intrahippocampal injection of the SK2 antagonist Lei-Dab⁷ before training impaired freezing when compared to non-treated mice. The same treatment showed no impairment when animals were pre-exposed for 5 min to the conditioning context 24 h before the training. Vehicle-treated animals were not different from non-treated animals (n = 7/group). (B) The effects of SK2 blockade before pre-exposure to the conditioning context on subsequent contextual fear conditioning in which 20 sec placement to shock interval is employed (CTX-S 20s). Injection of Lei-Dab⁷ before 5 min pre-exposure to context eliminated facilitation of the acquisition of context conditioning at a 20 sec placement to shock interval. Injection of vehicle before pre-exposure or 20 sec placement to shock training phase did not influence contextual pre-exposure effect (n = 8–9/group). (C) Lei-Dab⁷ was injected at the indicated time points after the training and before the memory test. Statistical comparison was made versus non-injected animals (n = 7–9/group). Arrows in the schematic experimental diagram indicate time points of Lei-Dab⁷ injection. Freezing was measured in the memory test 24 h after training. Data presented are the mean ± SEM. Statistics was performed by two-way ANOVA with Bonferroni multiple comparisons test (*<i>p</i> < 0.05).</p