Targeting Kca3.1 Channels in Cancer.

The Kca3.1 channels, previously designated as IK1 or SK4 channels and encoded by the KCNN4 gene, are activated by a rise of the intracellular Ca2+ concentration. These K+ channels are widely expressed in many organs and involved in many pathologies. In particular, Kca3.1 channels have been studied intensively in the context of cancer. They are not only a marker and a valid prognostic tool for cancer patients, but have an important share in driving cancer progression. Their function is required for many characteristic features of the aggressive cancer cell behavior such as migration, invasion and metastasis as well as proliferation and therapy resistance. In the context of cancer, another property of Kca3.1 is now emerging. These channels can be a target for novel small molecule-based imaging probes, as it has been validated in case of fluorescently labeled senicapoc-derivatives. The aim of this review is (i) to give an overview on the role of Kca3.1 channels in cancer progression and in shaping the cancer microenvironment, (ii) discuss the potential of using Kca3.1 targeting drugs for cancer imaging, (iii) and highlight the possibility of combining molecular dynamics simulations to image inhibitor binding to Kca3.1 channels in order to provide a deeper understanding of Kca3.1 channel pharmacology. Alltogether, Kca3.1 is an attractive therapeutic target so that senicapoc, originally developed for the treatment of sickle cell anemia, should be repurposed for the treatment of cancer patients.

PKA and Ube3a regulate SK2 channel trafficking to promote synaptic plasticity in hippocampus: Implications for Angelman Syndrome.

The ubiquitin ligase, Ube3a, plays important roles in brain development and functions, since its deficiency results in Angelman Syndrome (AS) while its over-expression increases the risk for autism. We previously showed that the lack of Ube3a-mediated ubiquitination of the Ca2+-activated small conductance potassium channel, SK2, contributes to impairment of synaptic plasticity and learning in AS mice. Synaptic SK2 levels are also regulated by protein kinase A (PKA), which phosphorylates SK2 in its C-terminal domain, facilitating its endocytosis. Here, we report that PKA activation restores theta burst stimulation (TBS)-induced long-term potentiation (LTP) in hippocampal slices from AS mice by enhancing SK2 internalization. While TBS-induced SK2 endocytosis is facilitated by PKA activation, SK2 recycling to synaptic membranes after TBS is inhibited by Ube3a. Molecular and cellular studies confirmed that phosphorylation of SK2 in the C-terminal domain increases its ubiquitination and endocytosis. Finally, PKA activation increases SK2 phosphorylation and ubiquitination in Ube3a-overexpressing mice. Our results indicate that, although both Ube3a-mediated ubiquitination and PKA-induced phosphorylation reduce synaptic SK2 levels, phosphorylation is mainly involved in TBS-induced endocytosis, while ubiquitination predominantly inhibits SK2 recycling. Understanding the complex interactions between PKA and Ube3a in the regulation of SK2 synaptic levels might provide new platforms for developing treatments for AS and various forms of autism.

Gain-of-Function Mutations in KCNN3 Encoding the Small-Conductance Ca2+-Activated K+ Channel SK3 Cause Zimmermann-Laband Syndrome.

Zimmermann-Laband syndrome (ZLS) is characterized by coarse facial features with gingival enlargement, intellectual disability (ID), hypertrichosis, and hypoplasia or aplasia of nails and terminal phalanges. De novo missense mutations in KCNH1 and KCNK4, encoding K+ channels, have been identified in subjects with ZLS and ZLS-like phenotype, respectively. We report de novo missense variants in KCNN3 in three individuals with typical clinical features of ZLS. KCNN3 (SK3/KCa2.3) constitutes one of three members of the small-conductance Ca2+-activated K+ (SK) channels that are part of a multiprotein complex consisting of the pore-forming channel subunits, the constitutively bound Ca2+ sensor calmodulin, protein kinase CK2, and protein phosphatase 2A. CK2 modulates Ca2+ sensitivity of the channels by phosphorylating SK-bound calmodulin. Patch-clamp whole-cell recordings of KCNN3 channel-expressing CHO cells demonstrated that disease-associated mutations result in gain of function of the mutant channels, characterized by increased Ca2+ sensitivity leading to faster and more complete activation of KCNN3 mutant channels. Pretreatment of cells with the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole revealed basal inhibition of wild-type and mutant KCNN3 channels by CK2. Analogous experiments with the KCNN3 p.Val450Leu mutant previously identified in a family with portal hypertension indicated basal constitutive channel activity and thus a different gain-of-function mechanism compared to the ZLS-associated mutant channels. With the report on de novo KCNK4 mutations in subjects with facial dysmorphism, hypertrichosis, epilepsy, ID, and gingival overgrowth, we propose to combine the phenotypes caused by mutations in KCNH1, KCNK4, and KCNN3 in a group of neurological potassium channelopathies caused by an increase in K+ conductance.

Unstructured to structured transition of an intrinsically disordered protein peptide in coupling Ca²âº-sensing and SK channel activation.

Most proteins, such as ion channels, form well-organized 3D structures to carry out their specific functions. A typical voltage-gated potassium channel subunit has six transmembrane segments (S1-S6) to form the voltage-sensing domain and the pore domain. Conformational changes of these domains result in opening of the channel pore. Intrinsically disordered (ID) proteins/peptides are considered equally important for the protein functions. However, it is difficult to explore the structural features underlying the functions of ID proteins/peptides by conventional methods, such as X-ray crystallography, because of the flexibility of their secondary structures. Unlike voltage-gated potassium channels, families of small- and intermediate-conductance Ca(2+)-activated potassium (SK/IK) channels with important roles in regulating membrane excitability are activated exclusively by Ca(2+)-bound calmodulin (CaM). Upon binding of Ca(2+) to CaM, a 2 × 2 structure forms between CaM and the CaM-binding domain. A channel fragment that connects S6 and the CaM-binding domain is not visible in the protein crystal structure, suggesting that this fragment is an ID fragment. Here we show that the conformation of the ID fragment in SK channels becomes readily identifiable in the presence of NS309, the most potent compound that potentiates the channel activities. This well-defined conformation of the ID fragment, stabilized by NS309, increases the channel open probability at a given Ca(2+) concentration. Our results demonstrate that the ID fragment, itself a target for drugs modulating SK channel activities, plays a unique role in coupling Ca(2+) sensing by CaM and mechanical opening of SK channels.

Protection against cardiac injury by small Ca(2+)-sensitive K(+) channels identified in guinea pig cardiac inner mitochondrial membrane.

We tested if small conductance, Ca(2+)-sensitive K(+) channels (SK(Ca)) precondition hearts against ischemia reperfusion (IR) injury by improving mitochondrial (m) bioenergetics, if O(2)-derived free radicals are required to initiate protection via SK(Ca) channels, and, importantly, if SK(Ca) channels are present in cardiac cell inner mitochondrial membrane (IMM). NADH and FAD, superoxide (O(2)(-)), and m[Ca(2+)] were measured in guinea pig isolated hearts by fluorescence spectrophotometry. SK(Ca) and IK(Ca) channel opener DCEBIO (DCEB) was given for 10 min and ended 20 min before IR. Either TBAP, a dismutator of O(2)()(-), NS8593, an antagonist of SK(Ca) isoforms, or other K(Ca) and K(ATP) channel antagonists, were given before DCEB and before ischemia. DCEB treatment resulted in a 2-fold increase in LV pressure on reperfusion and a 2.5 fold decrease in infarct size vs. non-treated hearts associated with reduced O(2)(-) and m[Ca(2+)], and more normalized NADH and FAD during IR. Only NS8593 and TBAP antagonized protection by DCEB. Localization of SK(Ca) channels to mitochondria and IMM was evidenced by a) identification of purified mSK(Ca) protein by Western blotting, immuno-histochemical staining, confocal microscopy, and immuno-gold electron microscopy, b) 2-D gel electrophoresis and mass spectroscopy of IMM protein, c) [Ca(2+)]-dependence of mSK(Ca) channels in planar lipid bilayers, and d) matrix K(+) influx induced by DCEB and blocked by SK(Ca) antagonist UCL1684. This study shows that 1) SK(Ca) channels are located and functional in IMM, 2) mSK(Ca) channel opening by DCEB leads to protection that is O(2)(-) dependent, and 3) protection by DCEB is evident beginning during ischemia.

Targeting SKCa channels in cancer: potential new therapeutic approaches.

Many studies have reported changes in potassium channel expression in many cancers and the involvement of these channels in various stages of cancer progression. By contrast, data concerning SKCa channels (small conductance calcium-activated potassium channels) have only recently become available. This review aims i) to present the structure and physiology of SKCa channels, ii) to provide an overview of published data concerning the SKCa proteins produced in tumor cells, and, whenever possible, the biological function assigned to them and iii) to review previous and novel modulators of SKCa channels. SKCa channels are activated by low concentrations of intracellular calcium and consist of homo- or heteromeric assemblies of α-subunits named SK1, SK2 and SK3. SK2-3 channels are expressed in tumors and have been assigned a biological function in cancer cells: the enhancement of cell proliferation and cell migration by hijacking the functions of SK2 and SK3 channels, respectively. Two major classes of SKCa modulators have been described: toxins (apamin) and small synthetic molecules. Most SKCa blockers are pore blockers, but some modify the calcium sensitivity of SKCa channels without interacting with the apamin binding site. In this review, we present edelfosine and ohmline as atypical anticancer agents and novel SK3 inhibitors. Edelfosine and ohmline are synthetic alkyl-lipids with structures different from all previously described SKCa modulators. They should pave the way for the development of a new class of migration-targeted anticancer agents. We believe that such blockers have potential for use in the prevention or treatment of metastasis.

Negative gating modulation by (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) depends on residues in the inner pore vestibule: pharmacological evidence of deep-pore gating of K(Ca)2 channels.

Acting as a negative gating modulator, (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) shifts the apparent Ca(2+)-dependence of the small-conductance Ca(2+)-activated K(+) channels K(Ca)2.1-2.3 to higher Ca(2+) concentrations. Similar to the positive K(Ca) channel-gating modulators 1-ethyl-2-benzimidazolinone (1-EBIO) and cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methylpyrimidin-4-yl]-amine (CyPPA), the binding site for NS8593 has been assumed to be located in the C-terminal region, in which these channels interact with their Ca(2+) sensor calmodulin. However, by using a progressive chimeric approach, we were able to localize the site-of-action of NS8593 to the K(Ca)2 pore. For example, when we transferred the C terminus from the NS8593-insensitive intermediate-conductance K(Ca)3.1 channel to K(Ca)2.3, the chimeric channel remained as sensitive to NS8593 as wild-type K(Ca)2.3. In contrast, when we transferred the K(Ca)2.3 pore to K(Ca)3.1, the channel became sensitive to NS8593. Using site-directed mutagenesis, we subsequently identified two specific residues in the inner vestibule of K(Ca)2.3 (Ser507 and Ala532) that determined the effect of NS8593. Mutation of these residues to the corresponding residues in K(Ca)3.1 (Thr250 and Val275) made K(Ca)2.3 insensitive to NS8593, whereas introduction of serine and alanine into K(Ca)3.1 was sufficient to render this channel highly sensitive to NS8593. It is noteworthy that the same two residue positions have been found previously to mediate sensitivity of K(Ca)3.1 to clotrimazole and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). The location of Ser507 in the pore-loop near the selectivity filter and Ala532 in an adjacent position in S6 are within the region predicted to contain the K(Ca)2 channel gate. Hence, we propose that NS8593-mediated gating modulation occurs via interaction with gating structures at a position deep within the inner pore vestibule.