SK Channels Research Articles

SK Channel Dysfunction in the Hypothalamic Paraventricular Nucleus Contributes to Sympathoexcitation in Dahl Salt‐Sensitive Rats

Salt‐sensitive hypertension is characterized by marked activation of the sympathetic nervous system. Previous studies have consistently demonstrated that increased excitability or activity of pre‐sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) contribute to the increased blood pressure and sympathetic nerve activity (SNA) in salt‐sensitive hypertension; however, the underlying mechanisms have not been fully elucidated. Small conductance calcium‐activated potassium (SK) channels act as negative feedback regulators of neuronal excitability in PVN neurons, and inhibition of SK channel function in the PVN is known to augment SNA and arterial blood pressure. In the present study, we tested the hypothesis that reduced SK channel function in the PVN contributes to sympathoexcitation in Dahl salt‐sensitive rats fed a high salt (HS, 4%) diet compared to a normal salt (NS, 0.4%) diet. Sympathoexcitatory responses to bilateral microinjection of the SK channel blocker apamin into the PVN were assessed in anesthetized 12–14‐week‐old male Dahl salt‐sensitive rats fed a high salt (n=7) or a normal salt (n=6) diet for 4 weeks. Baseline mean arterial pressure (MAP) was significantly elevated in rats fed a HS diet compared to NS (HS 129±4 vs. NS 114±4 mmHg, p<0.05), however baseline heart rate (HR) was not different between groups (HS 411±10 vs. NS 385±13 beats/min, p=0.07). Bilateral microinjection of apamin (12.5pmol, 50nL) into the PVN significantly increased splanchnic SNA in both HS and NS rats compared to baseline, however responses were significantly attenuated in HS rats compared to NS (HS 97±22% vs. NS 249±57%, p<0.01). In contrast, increases in MAP in response to PVN microinjection of apamin were similar between HS and NS rats (HS 24±4 vs. NS 29±4 mmHg, p=0.25), whereas HR responses were variable but not significantly different between groups (HS ‐8.5±9 vs. NS 7.5±7 beats/min, p=0.11). Next, we utilized qPCR to assess mRNA expression of SK1, SK2, and SK3 channels in PVN tissue from HS (n=5) and NS (n=6) rats. Interestingly, there was no significant difference in mRNA levels of either SK1, SK2, or SK3 channels between HS and NS fed rats. In summary, increases in splanchnic SNA due to PVN SK channel blockade with apamin were significantly attenuated in Dahl salt‐sensitive rats fed a HS diet, whereas there was no difference in SK channel expression between rats fed a HS vs. NS diet. These results suggest that dysfunction of SK channels in the PVN, but not decreased expression, contributes to sympathoexcitation Dahl salt‐sensitive rats fed a HS diet.

Repeated Restraint Stress Alters Small Conductance Calcium‐activated Potassium Channel (SK) Expression in Rat Basolateral Amygdala Neurons in Sex‐dependent Manner

Mood disorders disproportionally affect women more than men. The basolateral amygdala (BLA) is critical for the expression of fear and anxiety‐like behaviors and is hyperactive in mood disorders. Our prior work has shown greater spontaneous BLA activity in naïve females compared to males, but the mechanisms by which this occurs remain unknown. BLA activity is regulated, in part, by small conductance calcium‐activated potassium (SK) channels (SK1‐4), which mediate afterhyperpolarizations (AHPs) and reduce neuronal firing. Activation of the SK2 channel inhibits BLA neuronal firing in males, but the role of SK2 in females is relatively unexplored. We hypothesized that SK2 expression would be lower in females resulting in higher spontaneous firing rate of BLA neurons compared to males. In order to test this hypothesis, we utilized qPCR and Western blot analysis to determine the relative expression of SK channels in the BLA of adult naïve male and female rats. We found BLA SK2 mRNA levels are similar between the sexes, but BLA SK2 protein levels are lower in females compared to males. We found no differences in other SK channels. To examine whether lower SK2 expression in females corresponds with reduced SK2 channel activity, we performed in vivo single‐unit electrophysiological recordings of BLA neuronal activity following local injections of a SK2 channel inhibitor (apamin; 1uM) or saline. Preliminary results revealed spontaneous BLA activity increased following apamin injections in both sexes, but to a greater degree in males. This suggests that the reduced SK2 protein expression in females coincides with greater spontaneous activity in BLA and a diminished effect of apamin compared to males. Since it is known that stress is a leading factor in the development of mood disorders and BLA activity following stress also varies between the sexes; we also sought to determine whether sex differences exist in SK2 expression and function in rats following repeated restraint stress (20 min/day for 7 out of 9 days), compared to control‐handled rats. Previous research from our lab has found that repeated restraint stress leads to an increase in BLA spontaneous activity in males, but a reduction in activity in females. Therefore, we hypothesized that the expression and activity of SK2 channels will be reduced in males following stress, but increased in females. Our initial findings suggest that repeated restraint stress increases spontaneous BLA activity in males and this activity is less sensitive to apamin injection compared to control‐handled rats. In contrast, repeated restraint stress appears to reduce spontaneous BLA activity in females, but like the males, females have reduced sensitivity to apamin. These electrophysiological findings correspond to a reduction in SK2 protein expression in BLA from males, but a slight increase in SK2 expression in BLA from females. A more thorough understanding of sex differences in SK2 channel expression and activity following stress may contribute to a better understanding of the sex disparities observed in mood disorders.

PO-630-05 A NOVEL MECHANISM OF SMALL-CONDUCTANCE CA2+-ACTIVATED K+ CHANNEL ACTIVATION VIA NEURONAL NITRIC OXIDE SYNTHASE INHIBITION IN RAT ATRIA

The presence of atrial fibrillation (AF) is associated with electrical remodeling processes that promote a substrate for the maintenance of AF itself. Small-conductance Ca2+-activated K+ (SK) channel is a key factor in the atrial electrical remodeling. However, the mechanism of its activation is unclear. A recent study showed that neuronal nitric oxide synthase (nNOS) expression and activity are reduced in AF patients and that nNOS depletion causes the abbreviation of APD, leading to increased AF vulnerability in animals. Decreased NO production, especially driven by nNOS inhibition, might play a key role in the atrial electrical remodeling, and the downstream alteration of SK channels might result from this process. We aimed to evaluate the potential of SK channel blocking to mitigate abnormal electrophysiological properties and the inducibility of atrial tachyarrhythmia which was induced by neuronal nitric oxide synthase (nNOS) depletion, and to describe the related mechanism. Atrial tachyarrhythmia (ATA) induction and optical mapping were performed on perfused rat hearts. nNOS was pharmacologically inhibited by S-methylthiocitrulline (SMTC, 100nM). The influence of the SK channel was examined by a specific channel inhibitor, apamin (100nM). Action potential duration (APD), conduction velocity, and calcium transient (CaT) parameters (CaTD, Rise time, T 1/2, Tau) were evaluated by voltage and calcium optical mapping. Dominant frequency was examined in the analysis of AF dynamics. SMTC increased the inducibility of ATA and apamin mitigated the nNOS inhibition-induced arrhythmogenicity. SMTC caused the abbreviation and enhanced spatial dispersion of APD, which was reversed by apamin. In contrast, conduction velocity was not affected by SMTC or apamin. Moreover, apamin reduced the dominant frequency of SMTC-induced ATA. In voltage and calcium optical mapping, STMC and apamin does not altered parameters associated with CaT, however, SMTC caused abbreviation APD, which was reversed by apamin. Acute nNOS inhibition abbreviated APD via activating SK channels. A specific SK channel blocker mitigated APD abbreviation without alteration of CaT, implying an underlying mechanism of post translational modification of SK channels.

Vasodilators stimulate SK3 channel anterograde trafficking in endothelial cells to elicit vasodilation

Endothelial cells (ECs) line the lumen of blood vessels and regulate functions, including contractility. Physiological stimuli, such as acetylcholine (ACh) and intraluminal flow, activate small conductance calcium‐activated potassium (SK3) channels in ECs, leading to hyperpolarization and vasodilation. Whether these stimuli modulate SK3 surface abundance in ECs to elicit vasodilation is unclear. Here, we investigated physiological regulation of SK3 channel surface trafficking in mesenteric artery (MA) ECs. Biotinylation experiments demonstrated that ACh or intravascular flow rapidly (2 min) increased surface SK3 protein ~ 2.0‐ and 2.1‐fold, respectively in MAs, with similar results obtained in primary‐cultured ECs. Brefeldin A, an anterograde trafficking inhibitor, removal of extracellular Ca2+, GSK2193874, a selective TRPV4 channel blocker or TRPV4 knockdown using siRNA, each inhibited the ACh‐induced increase in surface SK3 channels in MAs. Super resolution microscopy (SMLM) experiments demonstrated that ACh increased the size of surface SK3 clusters in ECs. ACh activated whole‐cell SK currents and this effect was inhibited by brefeldin A. Brefeldin A also reduced vasodilation induced by ACh and intravascular flow to between ~48 and 57 % of those in control. Brefeldin A blocked the SK channel component of ACh‐ and flow‐mediated vasodilations. In summary, our data indicate that ACh and flow stimulate anterograde trafficking of SK3 channels in ECs via a TRPV4‐dependent Ca2+ signaling mechanism to elicit vasodilation.

PO-630-02 THE EFFECT OF CHANGES IN EXTRACELLULAR CALCIUM ON THE SMALL-CONDUCTION CA2+-ACTIVATED K+ CHANNEL OF METABOLIC SYNDROME

Metabolic syndrome (MetS) is associated with increased cardiac arrhythmia and mortality. Small conductance Ca2+-activated K+ (SK) channels link intracellular calcium transients to membrane potential changes and play an essential role in atrial repolarization. Regulation of the SK channels is still not fully discovered.

SKA-31-induced activation of small-conductance calcium-activated potassium channels decreased modulation of detrusor smooth muscle function in a rat model of obesity

Increased excitability and contractility of detrusor smooth muscle (DSM) cells are associated with overactive bladder (OAB), which is often induced by obesity. Small-conductance Ca2+-activated K+ (SK) channels regulate the excitability and contractility of DSM cells. Selective pharmacological activation of SK channels attenuates hyperpolarization and the decreased relaxation effect in DSM cells in obesity-induced OAB. However, additional data are needed to confirm the regulatory effect of SK channels on the function of DSM cells in obesity-related OAB. The tested hypothesis was that activation of SK channels decreases modulation of DSM function in a rat model of obesity-related OAB. Female Sprague Dawley rats were fed a normal diet (ND) or a high-fat diet (HFD), weighed after 12weeks, and subjected to urodynamic study, patch-clamp electrophysiology, and isometric tension recording. The average body weight and incidence of OAB were increased in the HFD group. Patch-clamp studies revealed that pharmacological activation of SK channels with SKA-31 had attenuated hyperpolarization of DSM cells. In addition, isometric tension recordings indicated that SKA-31 decreased relaxation of spontaneous phasic contractions of DSM strips in the HFD group. Attenuated function of SK channels increased the excitability and contractility of DSM cells, which contributed to the occurrence of OAB, suggesting that SK channels are potential therapeutic targets for control of OAB.

Antiarrhythmic Effects of Vernakalant in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes from a Patient with Short QT Syndrome Type 1.

(1) Background: Short QT syndrome (SQTS) may result in sudden cardiac death. So far, no drugs, except quinidine, have been demonstrated to be effective in some patients with SQTS type 1 (SQTS1). This study was designed to examine the potential effectiveness of vernakalant for treating SQTS1 patients, using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with SQTS1. (2) Methods: Patch clamp and calcium imaging techniques were used to examine the drug effects. (3) Results: Vernakalant prolonged the action potential duration (APD) in hiPSC-CMs from a SQTS1-patient (SQTS1-hiPSC-CMs). In spontaneously beating SQTS1-hiPSC-CMs, vernakalant reduced the arrhythmia-like events induced by carbachol plus epinephrine. Vernakalant failed to suppress the hERG channel currents but reduced the outward small-conductance calcium-activated potassium channel current. In addition, it enhanced Na/Ca exchanger currents and late sodium currents, in agreement with its APD-prolonging effect. (4) Conclusions: The results demonstrated that vernakalant can prolong APD and reduce arrhythmia-like events in SQTS1-hiPSC-CMs and may be a candidate drug for treating arrhythmias in SQTS1-patients.

Small conductance calcium-activated potassium channels and nitric oxide/cGMP pathway mediate cardioprotective effects of Croton urucurana Baill. In hypertensive rats

Ethnopharmacological relevanceCroton urucurana Baill. (Euphorbiaceae), popularly known as ‘sangue de dragão’ is a Brazilian species widely used in traditional medicine for cardiovascular ailments. AimTo investigate the cardiovascular effects of the C. urucurana extract in spontaneously hypertensive rats (SHRs). Materials and methodsLeaves from C. urucurana were collected and morphoanatomically characterized. The ethanol-soluble fraction (ESCU) was obtained and analyzed by LC-DAD-MS. Using female Wistar rats we investigated the acute toxicity of ESCU. Then, SHRs (six months old) received vehicle, hydrochlorothiazide (25 mg/kg), or ESCU (30, 100, 300 mg/kg) for 28 days. At the beginning and at the end of treatments, urine samples were obtained to assess renal function. At the end of the trial period, the blood pressure, mesenteric vascular beds (MVBs) reactivity, and electrocardiographic profile were evaluated. Serum angiotensin-converting enzyme activity, as well as urea, creatinine, sodium, potassium, nitrite, malondialdehyde, nitrotyrosine, and aldosterone levels were determined. Relative organ weights and histopathological analysis were performed. Finally, the cardiac function on a Langendorff system, as well as the molecular mechanisms involved in the vasodilator effects of ESCU in MVBs were also investigated. ResultsThe compounds annotated from ESCU by LC-DAD-MS included mainly phenylpropanoid derivatives, alkaloids, O-glycosylated megastigmanes, glycosylated flavonoids, flavan-3-ols, and others, such as quercetin O-deoxyhexosyl-hexoside, magnoflorine, reticuline, and taspine. None of the animals showed any signs of toxicity. Male SHRs treated only with the vehicle showed important cardiovascular changes, including a reduction in renal function, increase in serum oxidative stress, and hemodynamic, electrocardiographic, and morphological changes typical of hypertensive disease. Moreover, parameters of cardiac function, including left ventricular developed pressure, peak rate of contraction, peak rate of relaxation, and the rate pressure product were significantly altered, showing a significant impairment of ventricular function. All ESCU-doses presented a significant cardioprotective effect in SHRs rats. The 28-day treatment normalized the hemodynamic, electrocardiographic, morphological, and renal impairments, as well as reversed the changes in ventricular function induced by hypertension. In MVBs with an intact endothelium, ESCU (0.1, 0.3, and 1 mg) dose-dependently induced vasodilation. Endothelium removal or the inhibition of nitric oxide synthase prevented the vasodilatory effect of ESCU. Perfusion with a physiological saline solution that contained KCl, tetraethylammonium, or apamin also abolished the vasodilatory effect of ESCU. ConclusionProlonged ESCU-treatment showed significant cardioprotective effects in SHRs. Moreover, the data showed the role of nitric oxide and calcium-activated small conductance potassium channels in the cardiovascular effects of ESCU.

Role of detrusor PDGFR\u03b1+ cells in mouse model of cyclophosphamide-induced detrusor overactivity

Cyclophosphamide (CYP)-induced cystitis is a rodent model that shares many features common to the cystitis occurring in patients, including detrusor overactivity (DO). Platelet-derived growth factor receptor alpha positive (PDGFRα+) cells have been proposed to regulate muscle excitability in murine bladders during filling. PDGFRα+ cells express small conductance Ca2+-activated K+ channels (predominantly SK3) that provide stabilization of membrane potential during filling. We hypothesized that down-regulation of the regulatory functions of PDGFRα+ cells and/or loss of PDGFRα+ cells generates the DO in CYP-treated mice. After CYP treatment, transcripts of Pdgfrα and Kcnn3 and PDGFRα and SK3 protein were reduced in detrusor muscle extracts. The distribution of PDGFRα+ cells was also reduced. Inflammatory markers were increased in CYP-treated detrusor muscles. An SK channel agonist, CyPPA, increased outward current and hyperpolarization in PDGFRα+ cells. This response was significantly depressed in PDGFRα+ cells from CYP-treated bladders. Contractile experiments and ex vivo cystometry showed increased spontaneous contractions and transient contractions, respectively in CYP-treated bladders with a reduction of apamin sensitivity, that could be attributable to the reduction in the SK conductance expressed by PDGFRα+ cells. In summary, PDGFRα+ cells were reduced and the SK3 conductance was downregulated in CYP-treated bladders. These changes are consistent with the development of DO after CYP treatment.

The Inhibition of the Small-Conductance Ca2+-Activated Potassium Channels Decreases the Sinus Node Pacemaking during Beta-Adrenergic Activation.

Sinus pacemaking is based on tight cooperation of intracellular Ca2+ handling and surface membrane ion channels. An important player of this synergistic crosstalk could be the small-conductance Ca2+-activated K+-channel (ISK) that could contribute to the sinoatrial node (SAN) pacemaking driven by the intracellular Ca2+ changes under normal conditions and beta-adrenergic activation, however, the exact role is not fully clarified. SK2 channel expression was verified by immunoblot technique in rabbit SAN cells. Ionic currents and action potentials were measured by patch-clamp technique. The ECG R-R intervals were obtained by Langendorff-perfusion method on a rabbit heart. Apamin, a selective inhibitor of SK channels, was used during the experiments. Patch-clamp experiments revealed an apamin-sensitive current. When 100 nM apamin was applied, we found no change in the action potential nor in the ECG R-R interval. In experiments where isoproterenol was employed, apamin increased the cycle length of the SAN action potentials and enhanced the ECG R-R interval. Apamin did not amplify the cycle length variability or ECG R-R interval variability. Our data indicate that ISK has no role under normal condition, however, it moderately contributes to the SAN automaticity under beta-adrenergic activation.

A plasma membrane-localized polycystin-1/polycystin-2 complex in endothelial cells elicits vasodilation.

Polycystin-1 (PC-1, PKD1), a receptor-like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms, and physiological functions are poorly understood. Here, by studying tamoxifen-inducible, endothelial cell (EC)-specific Pkd1 knockout (Pkd1 ecKO) mice, we show that flow activates PC-1-mediated, Ca2+-dependent cation currents in ECs. EC-specific PC-1 knockout attenuates flow-mediated arterial hyperpolarization and vasodilation. PC-1-dependent vasodilation occurs over the entire functional shear stress range and via the activation of endothelial nitric oxide synthase (eNOS) and intermediate (IK)- and small (SK)-conductance Ca2+-activated K+ channels. EC-specific PC-1 knockout increases systemic blood pressure without altering kidney anatomy. PC-1 coimmunoprecipitates with polycystin-2 (PC-2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC-1 or PC-2 (Pkd2 ecKO mice) abolishes surface clusters of both PC-1 and PC-2 in ECs. Single knockout of PC-1 or PC-2 or double knockout of PC-1 and PC-2 (Pkd1/Pkd2 ecKO mice) similarly attenuates flow-mediated vasodilation. Flow stimulates nonselective cation currents in ECs that are similarly inhibited by either PC-1 or PC-2 knockout or by interference peptides corresponding to the C-terminus coiled-coil domains present in PC-1 or PC-2. In summary, we show that PC-1 regulates arterial contractility through the formation of an interdependent signaling complex with PC-2 in ECs. Flow stimulates PC-1/PC-2 clusters in the EC plasma membrane, leading to eNOS, IK channel, and SK channel activation, vasodilation, and a reduction in blood pressure.

Enhanced firing of locus coeruleus neurons and SK channel dysfunction are conserved in distinct models of prodromal Parkinson\u2019s disease

Parkinson’s disease (PD) is clinically defined by the presence of the cardinal motor symptoms, which are associated with a loss of dopaminergic nigrostriatal neurons in the substantia nigra pars compacta (SNpc). While SNpc neurons serve as the prototypical cell-type to study cellular vulnerability in PD, there is an unmet need to extent our efforts to other neurons at risk. The noradrenergic locus coeruleus (LC) represents one of the first brain structures affected in Parkinson’s disease (PD) and plays not only a crucial role for the evolving non-motor symptomatology, but it is also believed to contribute to disease progression by efferent noradrenergic deficiency. Therefore, we sought to characterize the electrophysiological properties of LC neurons in two distinct PD models: (1) in an in vivo mouse model of focal α-synuclein overexpression; and (2) in an in vitro rotenone-induced PD model. Despite the fundamental differences of these two PD models, α-synuclein overexpression as well as rotenone exposure led to an accelerated autonomous pacemaker frequency of LC neurons, accompanied by severe alterations of the afterhyperpolarization amplitude. On the mechanistic side, we suggest that Ca2+-activated K+ (SK) channels are mediators of the increased LC neuronal excitability, as pharmacological activation of these channels is sufficient to prevent increased LC pacemaking and subsequent neuronal loss in the LC following in vitro rotenone exposure. These findings suggest a role of SK channels in PD by linking α-synuclein- and rotenone-induced changes in LC firing rate to SK channel dysfunction.

Presynaptic NMDARs on spinal nociceptor terminals state-dependently modulate synaptic transmission and pain

Postsynaptic NMDARs at spinal synapses are required for postsynaptic long-term potentiation and chronic pain. However, how presynaptic NMDARs (PreNMDARs) in spinal nociceptor terminals control presynaptic plasticity and pain hypersensitivity has remained unclear. Here we report that PreNMDARs in spinal nociceptor terminals modulate synaptic transmission in a nociceptive tone-dependent manner. PreNMDARs depresses presynaptic transmission in basal state, while paradoxically causing presynaptic potentiation upon injury. This state-dependent modulation is dependent on Ca2+ influx via PreNMDARs. Small conductance Ca2+-activated K+ (SK) channels are responsible for PreNMDARs-mediated synaptic depression. Rather, tissue inflammation induces PreNMDARs-PKG-I-dependent BDNF secretion from spinal nociceptor terminals, leading to SK channels downregulation, which in turn converts presynaptic depression to potentiation. Our findings shed light on the state-dependent characteristics of PreNMDARs in spinal nociceptor terminals on modulating nociceptive transmission and revealed a mechanism underlying state-dependent transition. Moreover, we identify PreNMDARs in spinal nociceptor terminals as key constituents of activity-dependent pain sensitization.

Developing a stable full atom molecular dynamics model starting from a cyro-EM structure of a K+ channel bound to regulatory protein and varying PIP2 concentrations to understand regulation

Small conductance Ca2+-activated K+ (SK) channels are unique since they are gated solely by changes in intracellular Ca2+ and are known to be upregulated in heart failure and cardiac arrhythmias. SK channels are regulated via the ubiquitous Ca2+ sensing protein, calmodulin (CaM). New evidence suggests that the anionic lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is also essential for the activation for SK channels. Recent cryo-EM structures with a nearly complete tetrameric SK4-CaM complex (PDB: 6CNM, 6CNN, 6CNO) allowed us to generate nearly full-length SK2-CaM homology models (SK2-CaM).

SK channel enhancement reduces arrhythmogenesis in hereditary arrhythmia syndrome model with RyR2 complex gain-of-function

Small conductance Ca2+-activated K+ (SK) channels recently emerged having protective roles against Ca2+-dependent cardiac arrhythmias associated with cardiac hypertrophy and failure. This SK activation-mediated protection has been ascribed to increased repolarization reserve by SKs residing in the sarcolemma (sSK), and reduced mitochondrial production of reactive oxygen species (mito-ROS), given expression of SK channels in the inner mitochondrial membrane (mSK). We previously showed that mito-ROS reduction due to pharmacological facilitation of mSK activity stabilizes sarcoplasmic reticulum ryanodine receptor (RyR2) Ca2+ release, thus attenuating generation of proarrhythmic spontaneous Ca2+ waves.

Positive modulation of SKs channels by CyPPA depends on the HA/HB helices

In the activated state of small-conductance Ca2+-activated potassium (SK) channels, calmodulin interacts with the HA/HB helices and the S4-S5 linker. CyPPA potentiates SK2a and SK3 channel activity but not the SK1 and IK subtypes. Here, we report that the subtype-selectivity of CyPPA relies on the HA/HB helices. Mutating residues in the HA (V420) and HB (K467) helices of SK2a channels to their equivalent residues in IK channels diminished the potency of CyPPA. CyPPA elicited prominent responses on mutant IK channels with an arginine residue in the HB helix substituted for its equivalent lysine residue in the SK2a channels (R355K).

Histone deacetylase inhibitors (HDACi) increase expression of KCa2.3 (SK3) in primary microvascular endothelial cells.

The small conductance calcium-activated potassium channel (KCa2.3) has long been recognized for its role in mediating vasorelaxation through the endothelium-derived hyperpolarization (EDH) response. Histone deacetylases (HDACs) have been implicated as potential modulators of blood pressure and histone deacetylase inhibitors (HDACi) are being explored as therapeutics for hypertension. Herein, we show that HDACi increase KCa2.3 expression when heterologously expressed in HEK cells and endogenously expressed in primary cultures of human umbilical vein endothelial cells (HUVECs) and human intestinal microvascular endothelial cells (HIMECs). When primary endothelial cells were exposed to HDACi, KCa2.3 transcripts, subunits, and functional current are increased. Quantitative RT-PCR (qPCR) demonstrated increased KCa2.3 mRNA following HDACi, confirming transcriptional regulation of KCa2.3 by HDACs. By using pharmacological agents selective for different classes of HDACs, we discriminated between cytoplasmic and epigenetic modulation of KCa2.3. Biochemical analysis revealed an association between the cytoplasmic HDAC6 and KCa2.3 in immunoprecipitation studies. Specifically inhibiting HDAC6 increases expression of KCa2.3. In addition to increasing the expression of KCa2.3, we show that nonspecific inhibition of HDACs causes an increase in the expression of the molecular chaperone Hsp70 in endothelial cells. When Hsp70 is inhibited in the presence of HDACi, the magnitude of the increase in KCa2.3 expression is diminished. Finally, we show a slower rate of endocytosis of KCa2.3 as a result of exposure of primary endothelial cells to HDACi. These data provide the first demonstrated approach to increase KCa2.3 channel number in endothelial cells and may partially account for the mechanism by which HDACi induce vasorelaxation.

Electrophysiological Studies Support Utility of Positive Modulators of SK Channels for the Treatment of Spinocerebellar Ataxia Type 2.

Spinocerebellar ataxia type 2 (SCA2) is an incurable hereditary disorder accompanied by cerebellar degeneration following ataxic symptoms. The causative gene for SCA2 is ATXN2. The ataxin-2 protein is involved in RNA metabolism; the polyQ expansion may interrupt ataxin-2 interaction with its molecular targets, thus representing a loss-of-function mutation. However, mutant ataxin-2 protein also displays the features of gain-of-function mutation since it forms the aggregates in SCA2 cells and also enhances the IP3-induced calcium release in affected neurons. The cerebellar Purkinje cells (PCs) are primarily affected in SCA2. Their tonic pacemaker activity is crucial for the proper cerebellar functioning. Disturbances in PC pacemaking are observed in many ataxic disorders. The abnormal intrinsic pacemaking was reported in mouse models of episodic ataxia type 2 (EA2), SCA1, SCA2, SCA3, SCA6, Huntington's disease (HD), and in some other murine models of the disorders associated with the cerebellar degeneration. In our studies using SCA2-58Q transgenic mice via cerebellar slice recording and in vivo recording from urethane-anesthetized mice and awake head-fixed mice, we have demonstrated the impaired firing frequency and irregularity of PCs in these mice. PC pacemaker activity is regulated by SK channels. The pharmacological activation of SK channels has demonstrated some promising results in the electrophysiological experiments on EA2, SCA1, SCA2, SCA3, SCA6, HD mice, and also on mutant CACNA1A mice. In our studies, we have reported that the SK activators CyPPA and NS309 converted bursting activity into tonic, while oral treatment with CyPPA and NS13001 significantly improved motor performance and PC morphology in SCA2 mice. The i.p. injections of chlorzoxazone (CHZ) during in vivo recording sessions converted bursting cells into tonic in anesthetized SCA2 mice. And, finally, long-term injections of CHZ recovered the precision of PC pacemaking activity in awake SCA2 mice and alleviated their motor decline. Thus, the SK activation can be used as a potential way to treat SCA2 and other diseases accompanied by cerebellar degeneration.

Antidepressant activity of pharmacological and genetic deactivation of the small-conductance calcium-activated potassium channel subtype-3.

The voltage-insensitive, small-conductance calcium-activated potassium (SK) channel is a key regulator of neuronal depolarization and is implicated in the pathophysiology of depressive disorders. We ascertained whether the SK channel is impaired in the chronic unpredictable stress (CUS) model and whether it can serve as a molecular target of antidepressant action. We assessed the depressive-like behavioral phenotype of CUS-exposed rats and performed post-mortem SK channel binding and activity-dependent zif268 mRNA analyses on their brains. To begin an assessment of SK channel subtypes involved, we examined the effects of genetic and pharmacological inhibition of the SK3 channel using conditional knockout mice and selective SK3 channel negative allosteric modulators (NAMs). We found that [125I]apamin binding to SK channels is increased in the prefrontal cortex and decreased in the hippocampus, an effect that was associated with reciprocal levels of zif268 mRNA transcripts indicating abnormal regional cell activity in this model. We found that genetic and pharmacological manipulations significantly decreased immobility in the forced swim test without altering general locomotor activity, a hallmark of antidepressant-like activity. Taken together, these findings link depression-related neural and behavioral pathophysiology with abnormal SK channel functioning and suggest that this can be reversed by the selective inhibition of SK3 channels.

Reduction of neuronal hyperexcitability with modulation of T-type calcium channel or SK channel in essential tremor.

Essential tremor is one of the most prevalent movement disorders. Propranolol and primidone are the first-line pharmacological therapies. They provide symptomatic control in less than 50% of patients. Topiramate, alprazolam, clonazepam, gabapentin, and botulinum toxin injections are the next line of treatments. These medications lead to modest improvements and are therefore commonly used as add-on agents. Surgical therapies, including deep brain stimulation (DBS) surgery and focused ultrasound beam targeted to the thalamus, are considered for treating tremor refractory to medications and lead to greater than 75% improvements in tremor symptoms. However, DBS is a costly and an invasive procedure; some patients report tolerance to benefits. Focused ultrasound therapy leading to brain lesions is associated with a possibility for permanent clinical deficits. Therefore, research efforts to develop the next generation of oral medications with greater benefits and lesser adverse effects are warranted. There is considerable evidence that the increased functions of calcium channels (P/Q-type and T-type channels) and reduced functions of calcium-activated potassium channels (SK channels) located in the neuronal membranes lead to tremor oscillations. Consequently, many new pharmacological studies have targeted these channels to leverage better clinical outcomes. The current review will discuss the pathophysiology, the specific importance of these channels, and the early clinical experience of using compounds targeting these channels to treat essential tremor.