EF hands at the N-lobe of calmodulin are required for both SK channel gating and stable SK–calmodulin interaction

Small conductance calcium-activated potassium (SK) channels respond to intracellular Ca²⁺ via constitutively associated calmodulin (CaM). Previous studies have proposed a modular design for the interaction between CaM and SK channels. The C-lobe and the linker of CaM are thought to regulate the constitutive binding, whereas the N-lobe binds Ca²⁺ and gates SK channels. However, we found that coexpression of mutant CaM (E/Q) where the N-lobe has only one functional EF hand leads to rapid rundown of SK channel activity, which can be recovered with exogenously applied wild-type (WT), but not mutant, CaM. Our results suggest that the mutation at the N-lobe EF hand disrupts the stable interaction between CaM and SK channel subunits, such that mutant CaM dissociates from the channel complex when the inside of the membrane is exposed to CaM-free solution. The disruption of the stable interaction does not directly result from the loss of Ca²⁺-binding capacity because SK channels and WT CaM can stably interact in the absence of Ca²⁺. These findings question a previous conclusion that CaM where the N-lobe has only one functional EF hand can stably support the gating of SK channels. They cannot be explained by the current model of modular interaction between CaM and SK channels, and they imply a role for N-lobe EF hand residues in binding to the channel subunits. Additionally, we found that a potent enhancer for SK channels, 3-oxime-6,7-dichloro-1H-indole-2,3-dione (NS309), enables the recovery of channel activity with CaM (E/Q), suggesting that NS309 stabilizes the interaction between CaM and SK channels. CaM (E/Q) can regulate Ca²⁺-dependent gating of SK channels in the presence of NS309, but with a lower apparent Ca²⁺ affinity than WT CaM.     

Chemical modifications of the N-methyl-laudanosine scaffold point to new directions for SK channels exploration

An asparagine or a histidine are present in a similar position in the outer pore region of SK2 and SK3 channels, respectively. Therefore, this structural difference was targeted in order to develop selective blockers of SK channel subtypes. Following docking investigations, based on theoretical models of truncated SK2 and SK3 channels, the benzyl side chain of N-methyl-laudanosine (NML) was functionalized in order to target this specific amino-acid residues. Chiral butanamide and benzyloxy analogues were prepared, resolved and tested for their affinity for SK2 and SK3 channels. Isoquinolinium (NMIQ) derivatives have a higher affinity for both SK channel subtypes than the corresponding derivative with no functionalized side chain. This trend was observed also for the 1,2,3,4-tetrahydroisoquinoline (THIQ) analogues. A benzyloxy functionalized NML enantiomer has a higher affinity than NML stereoisomers. Otherwise, the conserved affinity of these analogues led to the opportunity to further investigate in terms of possible labeling for in vivo investigations of the role of SK channels.     

The SK3 subunit of small conductance Ca2+-activated K+ channels interacts with both SK1 and SK2 subunits in a heterologous expression system

The aim of this study was to determine whether functional heteromeric channels can be formed by co-assembly of rat SK3 (rSK3) potassium channel subunits with either SK1 or SK2 subunits. First, to determine whether rSK3 could co-assemble with rSK2 we created rSK3VK (an SK3 mutant insensitive to block by UCL 1848). When rSK3VK was co-expressed with rSK2 the resulting currents had an intermediate sensitivity to UCL 1848 (IC50 of approximately 5 nM compared with 120 pM for rSK2 and >300 nM for rSK3VK), suggesting that rSK3 and rSK2 can form functional heteromeric channels. To detect co-assembly of SK3 with SK1, we initially used a dominant negative construct of the human SK1 subunit (hSK1YP). hSK1YP dramatically reduced the SK3 current, supporting the idea that SK3 and SK1 subunits also interact. To determine whether these assemblies were functional we created rSK3VF, an rSK3 mutant with an enhanced affinity for tetraethylammonium chloride (TEA) (IC50 of 0.3 mM). Co-transfection of rSK3VF and hSK1 produced currents with a sensitivity to TEA not different from that of hSK1 alone (IC50 approximately 15 mM). These results suggest that hSK1 does not produce functional cell-surface assemblies with SK3. Antibody-staining experiments suggested that hSK1 may reduce the number of functional SK3 subunits reaching the cell surface. Additional experiments showed that co-expression of the rat SK1 gene with SK3 also dramatically suppressed SK current. The pharmacology of the residual current was consistent with that of homomeric SK3 assemblies. These results demonstrate interactions that cause changes in protein trafficking, cell surface expression, and channel pharmacology and strongly suggest heteromeric assembly of SK3 with the other SK channel subunits.     

SK but not IK channels regulate human detrusor smooth muscle spontaneous and nerve-evoked contractions

Animal studies suggest that the small (SK) and intermediate (IK) conductance Ca(2+)-activated K(+) channels may contribute to detrusor smooth muscle (DSM) excitability and contractility. However, the ability of SK and IK channels to control DSM spontaneous phasic and nerve-evoked contractions in human DSM remains unclear. We first investigated SK and IK channels molecular expression in native human DSM and further assessed their functional role using isometric DSM tension recordings and SK/IK channel-selective inhibitors. Quantitative PCR experiments revealed that SK3 channel mRNA expression in isolated DSM single cells was ～12- to 44-fold higher than SK1, SK2, and IK channels. RT-PCR studies at the single-cell level detected mRNA messages for SK3 channels but not SK1, SK2, and IK channels. Western blot and immunohistochemistry analysis further confirmed protein expression for the SK3 channel and lack of detectable protein expression for IK channel in whole DSM tissue. Apamin (1 μM), a selective SK channel inhibitor, significantly increased the spontaneous phasic contraction amplitude, muscle force integral, phasic contraction duration, and muscle tone of human DSM isolated strips. Apamin (1 μM) also increased the amplitude of human DSM electrical field stimulation (EFS)-induced contractions. However, TRAM-34 (1 μM), a selective IK channel inhibitor, had no effect on the spontaneous phasic and EFS-induced DSM contractions suggesting a lack of IK channel functional role in human DSM. In summary, our molecular and functional studies revealed that the SK, particularly the SK3 subtype, but not IK channels are expressed and regulate the spontaneous and nerve-evoked contractions in human DSM.     

Synthesis and biological evaluation of N-methyl-laudanosine iodide analogues as potential SK channel blockers

Neuronal action potentials are followed by an afterhyperpolarisation (AHP), which is mediated by small conductance Ca2+-activated K+ channels (SK channels or KCa2 channels). This AHP plays an important role in regulating neuronal activity and agents modulating AHP amplitude could have a potential therapeutic interest. It was previously shown that N-methyl-bicuculline iodide blocks SK channels but its GABA) activity represents a serious drawback. In view of the structural analogy between bicuculline and laudanosine 14, several N-quaternary analogues of the latter were developed. It was shown that N-methyl-laudanosine 15 (NML) and N-ethyl-laudanosine 16 induce a reversible and relatively specific blockade of the apamin sensitive AHP in dopaminergic neurones with mean IC50s of 15, and 47 microM, respectively. Laudanosine 14, N-butyl-17 and N-benzyl-18 derivatives were less potent. In order to find pharmacophore elements, modifications were performed at different positions such as C-1, C-6 and C-7. Intracellular recordings on rat midbrain dopaminergic neurones were made in order to evaluate the putative blockade of SK channels by these molecules. Simplified structures such as tetrahydroisoquinoline derivatives with H or Me at C-1 1-6 presented no significant activity at 300 microM. The presence of a 1-(3,4-dimethoxybenzyl) moiety seems an important feature. Indeed, compound 8 showed a blockade of the AHP of only 33% at 300 microM while compound 13 blocked it by 67%, respectively, at the same concentration. Binding experiments were also performed. Binding affinities for SK channels are in good agreement with electrophysiological data. These results indicate that the presence of a charged nitrogen group is an essential point for the affinity on SK channels. Finally, because of the similar activity of both enantiomers of NML 19 and 20, the interaction site may present a symmetrical configuration.     

Domain analysis of the calcium-activated potassium channel SK1 from rat brain. Functional expression and toxin sensitivity

Two small conductance, calcium-activated potassium channels (SK channels), SK2 and SK3, have been shown to contribute to the afterhyperpolarization (AHP) and to shape the firing behavior in neurons for example in the hippocampal formation, the dorsal vagal nucleus, the subthalamic nucleus, and the cerebellum. In heterologous expression systems, SK2 and SK3 currents are blocked by the bee venom toxin apamin, just as well as the corresponding neuronal AHP currents. However, the functional role and pharmacological profile of SK1 channels from rat brain (rSK1) is still largely unknown, as so far rSK1 homomeric channels could not be functionally expressed. We have performed a domain analysis to elucidate the pharmacological profile and the molecular determinants of rSK1 channel expression by using channel chimeras in combination with immunocytochemistry, immunoblot analysis, and electrophysiology. Our results reveal that the rSK1 subunit is synthesized in cells but does not form functional homomeric channels. Exchanging the carboxyl terminus of rSK1 for that of hSK1 or rSK2 is sufficient to rescue the functional expression of rSK1 channels. Additionally, transplantation of both amino and carboxyl termini of rSK1 onto hSK1 subunits, normally forming functional homomeric channel, hinders their functional expression, while hSK1 channels containing only the rSK1 carboxyl terminus are functional. These results suggest that the lack of functional expression of rSK1 channels is probably due to problems in their assembly and tetramerization but not in their calmodulin-dependent gating. Finally, we show that chimeric channels containing the core domain (S1-S6) of rSK1, unlike hSK1, are apamin-insensitive.     

A novel isoform of SK2 assembles with other SK subunits in mouse brain

The SK2 subtype of small conductance Ca2+-activated K+ channels is widely distributed throughout the central nervous system and modulates neuronal excitability by contributing to the afterhyperpolarization that follows an action potential. Western blots of brain membrane proteins prepared from wild type and SK2-null mice reveal two isoforms of SK2, a 49-kDa band corresponding to the previously reported SK2 protein (SK2-S) and a novel 78-kDa form. Complementary DNA clones from brain and Western blots probed with an antibody specific for the longer form, SK2-L, identified the larger molecular weight isoform as an N-terminally extended SK2 protein. The N-terminal extension of SK2-L is cysteine-rich and mediates disulfide bond formation between SK2-L subunits or with heterologous proteins. Immunohistochemistry revealed that in brain SK2-L and SK2-S are expressed in similar but not identical patterns. Heterologous expression of SK2-L results in functional homomeric channels with Ca2+ sensitivity similar to that of SK2-S, consistent with their shared core and intracellular C-terminal domains. In contrast to the diffuse, uniform surface distribution of SK2-S, SK2-L channels cluster into sharply defined, distinct puncta suggesting that the extended cysteine-rich N-terminal domain mediates this process. Immunoprecipitations from transfected cells and mouse brain demonstrate that SK2-L co-assembles with the other SK subunits. Taken together, the results show that the SK2 gene encodes two subunit proteins and suggest that native SK2-L subunits may preferentially partition into heteromeric channel complexes with other SK subunits.     

Decreased expression of small-conductance Ca2+-activated K+ channels SK1 and SK2 in human chronic atrial fibrillation

AimsSmall-conductance Ca^2 +-activated K⁺ (SK) channels are recognized as new ion channel candidates in atrial fibrillation (AF), with pivotal implications as novel drug targets due to their atrial-selective distribution in humans. The purpose of this study was to investigate whether SK channels and the Ca^2 +-activated K⁺ current (I_K,Ca) are involved in electrical remodeling of human chronic AF (cAF) and whether they display the differential distribution between the right (RA) and left atria (LA).Main methodsThe right (RAA) and left atrial appendage (LAA) myocytes were obtained from 29 sinus rhythm (SR) and 22 cAF patients. The I_K,Ca and action potential (AP) were recorded using the patch-clamp technique. Three SK channel subtypes (SK1–3) expressions were assayed by western blot and real-time quantitative PCR analysis.Key findingsThe I_K,Ca was decreased and its role in AP repolarization was attenuated in cAF, concomitant with a significant decrease in protein and mRNA levels of SK1 and SK2. In either SR or cAF, there was no difference in the I_K,Ca density and protein and mRNA expression levels of SK1–3 between RAA and LAA myocytes.SignificanceOur results demonstrated that SK1 and SK2 are involved in electrical remodeling of cAF. SK1–3 and I_K,Ca do not display the inter-atrial differential distribution in SR or cAF. These findings provide a new insight into mechanisms of electrical remodeling of human cAF.     

Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons

Small-conductance Ca²⁺-activated K⁺ channel activation is an emerging therapeutic approach for treatment of neurological diseases, including stroke, amyotrophic lateral sclerosis and schizophrenia. Our previous studies showed that activation of SK channels exerted neuroprotective effects through inhibition of NMDAR-mediated excitotoxicity. In this study, we tested the therapeutic potential of SK channel activation of NS309 (25 μM) in cultured human postmitotic dopaminergic neurons in vitro conditionally immortalized and differentiated from human fetal mesencephalic cells. Quantitative RT-PCR and western blotting analysis showed that differentiated dopaminergic neurons expressed low levels of SK2 channels and high levels of SK1 and SK3 channels. Further, protein analysis of subcellular fractions revealed expression of SK2 channel subtype in mitochondrial-enriched fraction. Mitochondrial complex I inhibitor rotenone (0.5 μM) disrupted the dendritic network of human dopaminergic neurons and induced neuronal death. SK channel activation reduced mitochondrial membrane potential, while it preserved the dendritic network, cell viability and ATP levels after rotenone challenge. Mitochondrial dysfunction and delayed dopaminergic cell death were prevented by increasing and/or stabilizing SK channel activity. Overall, our findings show that activation of SK channels provides protective effects in human dopaminergic neurons, likely via activation of both membrane and mitochondrial SK channels. Thus, SK channels are promising therapeutic targets for neurodegenerative disorders such as Parkinson''s disease, where dopaminergic cell loss is associated with progression of the disease.     

Functions and Modulation of Neuronal SK Channels

Small conductance (SK) channels are calcium-activated potassium channels that, when cloned in 1996, were thought solely to contribute to the afterhyperpolarisation that follows action potentials, and to control repetitive firing patterns of neurons. However, discoveries over the past few years have identified novel roles for SK channels in controlling dendritic excitability, synaptic transmission and synaptic plasticity. More recently, modulation of SK channel calcium sensitivity by casein kinase 2, and of SK channel trafficking by protein kinase A, have been demonstrated. This article will discuss recent findings regarding the function and modulation of SK channels in central neurons.     

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

Small conductance Ca²⁺-sensitive potassium (SK2) channels are voltage-independent, Ca²⁺-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca²⁺ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca²⁺ and Ca²⁺-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca²⁺ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.     

Cloning and functional expression of a liver isoform of the small conductance Ca2+-activated K+ channel SK3

Small conductance Ca2+-activated K+ (SK) channels have been cloned from mammalian brain, but little is known about the molecular characteristics of SK channels in nonexcitable tissues. Here, we report the isolation from rat liver of an isoform of SK3. The sequence of the rat liver isoform differs from rat brain SK3 in five amino acid residues in the NH3 terminus, where it more closely resembles human brain SK3. SK3 immunoreactivity was detectable in hepatocytes in rat liver and in HTC rat hepatoma cells. Human embryonic kidney (HEK-293) cells transfected with liver SK3 expressed 10 pS K+ channels that were Ca2+ dependent (EC(50) 630 nM) and were blocked by the SK channel inhibitor apamin (IC(50) 0.6 nM); whole cell SK3 currents inactivated at membrane potentials more positive than -40 mV. Notably, the Ca2+ dependence, apamin sensitivity, and voltage-dependent inactivation of SK3 are strikingly similar to the properties of hepatocellular and biliary epithelial SK channels evoked by metabolic stress. These observations raise the possibility that SK3 channels influence membrane K+ permeability in hepatobiliary cells during liver injury.     

Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

Small conductance Ca²⁺-sensitive potassium (SK2) channels are voltage-independent, Ca²⁺-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca²⁺ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca²⁺ and Ca²⁺-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca²⁺ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.     

Functional interactions between the SK2 channel and the nicotinic acetylcholine receptor in enteric neurons of the guinea pig ileum

The neurotransmitter acetylcholine (ACh) plays a critical role in gastrointestinal function. The role of the small conductance Ca²⁺-activated K⁺ (SK) channel in ACh release was examined using myenteric plexus preparations of guinea pig ileum. Apamin, an inhibitor of the SK channel, significantly enhanced nicotine-induced ACh release, but neither electrical field stimulation- nor 5-hydroxytryptamine-induced ACh release, suggesting that SK channels might be selectively involved in the regulation of nicotine-induced ACh release. Therefore, we investigated the distribution of SK2 and SK3 subunits and the interaction between SK2 channels and nicotinic ACh receptors (nAChRs) in the guinea pig ileum. The immunoreactivity of SK2 subunits was located in enteric neuronal cells. Furthermore, SK2-immunoreactive cells stained with an antibody for choline acetyltransferase, a marker for cholinergic neurons, and with an antibody for the α3/5 subunits of nAChR. In contrast, immunoreactivity of SK3 subunits was not found in enteric neurons. A co-immunoprecipitation assay with Triton X-100-soluble membrane fractions prepared from the ileum revealed an association of the SK2 subunit with the α3/5 subunits of nAChR. These results suggest that SK2 channels negatively regulate the excitation of enteric neurons via functional interactions with nAChRs.     

Subtype-specific, bi-component inhibition of SK channels by low internal pH

The effects of low intracellular pH (pH(i) 6.4) on cloned small-conductance Ca2+-activated K+ channel currents of all three subtypes (SK1, SK2, and SK3) were investigated in HEK293 cells using the patch-clamp technique. In 400 nM internal Ca2+ [Ca2+]i, all subtypes were inhibited by pH(i) 6.4 in the order of sensitivity: SK1>SK3>SK2. The inhibition increased with the transmembrane voltage. In saturating internal Ca2+, the inhibition was abolished for SK1-3 channels at negative potentials, indicating a [Ca2+]i-dependent mode of inhibition. Application of 50 microM 1-ethyl-2-benzimidazolone was able to potentiate SK3 current to the same extent as at neutral pH(i). We conclude that SK1-3 all are inhibited by low pH(i). We suggest two components of inhibition: a [Ca2+]i-dependent component, likely involving the SK beta-subunits calmodulin, and a voltage-dependent component, consistent with a pore-blocking effect. This pH(i)-dependent inhibition can be reversed pharmacologically.     

Metformin regulates atrial SK2 and SK3 expression through inhibiting the PKC/ERK signaling pathway in type 2 diabetic rats

Background

Our previous study showed that metformin regulates the mRNA and protein levels of type 2 small conductance calcium-activated potassium channel (SK2) and type 3 small conductance calcium-activated potassium channels (SK3) in atrial tissue as well as the ion current of atrial myocytes in rats with type 2 diabetes mellitus (T2DM), but the underlying signaling mechanism is unknown. This study aimed to investigate whether metformin regulates atrial SK2 and SK3 protein expression in T2DM rats though the protein kinase C (PKC)/extracellular signal-regulated kinase (ERK) signaling pathway.

Methods

A T2DM rat model was established using a high-fat and high-sugar diet combined with a low-dose intraperitoneal injection of streptozotocin (STZ). The rats were randomly divided into the following five groups: the control group, the untreated T2DM group, the metformin-treated only group, the phorbol 12-myristate 13-acetate (PMA; a PKC agonist administered by intraperitoneal injection) treatment group, and the recombinant human epidermal growth factor (rh-EGF; an ERK agonist administered by tail vein injection) treatment group. The activity of PKC in atrial tissues was assayed by a PKC kinase activity assay kit. The protein expression of SK2, SK3, and phosphorylated ERK (pERK) were determined by western blotting and immunohistochemistry.

Results

Compared with the Control group, atrial PKC activity and pERK and SK3 protein expression were increased, while SK2 protein expression was decreased in atrial tissues of T2DM rats. Eight weeks of metformin treatment inhibited the PKC activity and pERK and SK3 expression, and elevated SK2 expression compared with the T2DM group. Compared with the metformin-treated only group, the injection of rh-EGF increased pERK and SK3 expression, and decreased SK2 expression; the injection of PMA increased PKC activity and SK3 expression, and decreased SK2 expression. In addition, the injection with PMA significantly elevated the expression of pERK.

Conclusions

The PKC/ERK signaling pathway is involved in the downregulation of SK2 expression and the upregulation of SK3 expression in the atrium of T2DM rats. Long-term metformin treatment prevents the SK2 downregulation and the SK3 upregulation through inhibiting the PKC/ERK signaling pathway.

     

Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels

This study examines the roles of voltage-dependent Ca(2+) channels (VDCC), ryanodine receptors (RyRs), large-conductance Ca(2+)-activated K(+) (BK) channels, and small-conductance Ca(2+)-activated K(+) (SK) channels in the regulation of phasic contractions of guinea pig urinary bladder smooth muscle (UBSM). Nisoldipine (100 nM), a dihydropyridine inhibitor of VDCC, abolished spontaneous UBSM contractions. Ryanodine (10 microM) increased contraction frequency and thereby integrated force and, in the presence of the SK blocker apamin, had a greater effect on integrated force than ryanodine alone. Blocking BK (iberiotoxin, 100 nM) or SK (apamin, 100 nM) channels increased contraction amplitude and duration but decreased frequency. The contractile response to iberiotoxin was more pronounced than to apamin. The increases in contraction amplitude and duration to apamin were substantially augmented with ryanodine pretreatment. These results indicate that BK and SK channels have prominent roles as negative feedback elements to limit UBSM contraction amplitude and duration. RyRs also appear to play a significant role as a negative feedback regulator of contraction frequency and duration, and this role is influenced by the activity of SK channels.