Molecular overlap in the regulation of SK channels by small molecules and phosphoinositides

Phosphatidylinositol 4,5-bisphosphate (PIP2) directly interacts with the small-conductance Ca2+-activated K+ 2-a (SK2-a) channel/calmodulin complex, serving as a critical element in the regulation of channel activity. We report that changes of protein conformation in close proximity to the PIP2 binding site induced by a small-molecule SK channel modulator, NS309, can effectively enhance the interaction between the protein and PIP2 to potentiate channel activity. This novel modulation of PIP2 sensitivity by small-molecule drugs is likely not to be limited in its application to SK channels, representing an intriguing strategy to develop drugs controlling the activity of the large number of PIP2-dependent proteins.

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

The Journal of General Physiology ◽

10.1085/jgp.200910295 ◽

2009 ◽

Vol 134 (4) ◽

pp. 281-293 ◽

Cited By ~ 44

Author(s):

Weiyan Li ◽

David B. Halling ◽

Amelia W. Hall ◽

Richard W. Aldrich

Keyword(s):

Modular Design ◽

Binding Capacity ◽

Current Model ◽

Sk Channels ◽

Channel Activity ◽

Wild Type ◽

Sk Channel ◽

Ef Hand ◽

Ef Hands ◽

Small Conductance

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.

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Mineralocorticoids decrease the activity of the apical small-conductance K channel in the cortical collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.00063.2005 ◽

2005 ◽

Vol 289 (5) ◽

pp. F1065-F1071 ◽

Cited By ~ 7

Author(s):

Yuan Wei ◽

Elisa Babilonia ◽

Hyacinth Sterling ◽

Yan Jin ◽

Wen-Hui Wang

Keyword(s):

Protein Tyrosine Kinase ◽

Collecting Duct ◽

K Channel ◽

Sk Channels ◽

Cortical Collecting Duct ◽

Channel Activity ◽

Patch Clamp Technique ◽

Herbimycin A ◽

Sk Channel ◽

Small Conductance

We used the patch-clamp technique to examine the effect of DOCA treatment (2 mg/kg) on the apical small-conductance K (SK) channels, epithelial Na channels (ENaC), and the basolateral 18-pS K channels in the cortical collecting duct (CCD). Treatment of rats with DOCA for 6 days significantly decreased the plasma K from 3.8 to 3.1 meq and reduced the activity of the SK channel, defined as NPo, from 1.3 in the CCD of control rats to 0.6. In contrast, DOCA treatment significantly increased ENaC activity from 0.01 to 0.53 and the basolateral 18-pS K channel activity from 0.67 to 1.63. Moreover, Western blot analysis revealed that DOCA treatment significantly increased the expression of the nonreceptor type of protein tyrosine kinase (PTK), cSrc, and the tyrosine phosphorylation of ROMK in the renal cortex and outer medulla. The possibility that decreases in apical SK channel activity induced by DOCA treatment were the result of stimulation of PTK activity was further supported by experiments in which inhibition of PTK with herbimycin A significantly increased NPo from 0.6 to 2.1 in the CCD from rats receiving DOCA. Also, when rats were fed a high-K (10%) diet, DOCA treatment did not increase the expression of c-Src and decrease the activity of the SK channel in the CCD. We conclude that DOCA treatment decreased the apical SK channel activity in rats on a normal-K diet and that an increase in PTK expression may be responsible for decreased channel activity in the CCD from DOCA-treated rats.

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Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD

AJP Renal Physiology ◽

10.1152/ajprenal.00352.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. F806-F812 ◽

Cited By ~ 8

Author(s):

Dimin Li ◽

Yuan Wei ◽

Elisa Babilonia ◽

Zhijian Wang ◽

Wen-Hui Wang

Keyword(s):

Collecting Duct ◽

Sk Channels ◽

Channel Activity ◽

Phosphatidylinositol 3 Kinase ◽

Patch Clamp Technique ◽

Α Subunit ◽

Sk Channel ◽

Low K ◽

Small Conductance ◽

Phosphatidylinositol 3

We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa α-subunit (p110α) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels ( 2 ), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H2O2 stimulates the expression of p110α in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton.

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Hydrolyzable ATP and PIP2 Modulate the Small-conductance K+ Channel in Apical Membranes of Rat Cortical-Collecting Duct (CCD)

The Journal of General Physiology ◽

10.1085/jgp.20028677 ◽

2002 ◽

Vol 120 (5) ◽

pp. 603-615 ◽

Cited By ~ 19

Author(s):

Ming Lu ◽

Steven C. Hebert ◽

Gerhard Giebisch

Keyword(s):

Kinase Inhibitors ◽

Collecting Duct ◽

K Channel ◽

Sk Channels ◽

Cortical Collecting Duct ◽

Channel Activity ◽

Sk Channel ◽

Phosphoinositide Kinase ◽

Excised Patches ◽

Small Conductance

The small-conductance K+ channel (SK) in the apical membrane of the cortical-collecting duct (CCD) is regulated by adenosine triphosphate (ATP) and phosphorylation-dephosphorylation processes. When expressed in Xenopus oocytes, ROMK, a cloned K+ channel similar to the native SK channel, can be stimulated by phosphatidylinositol bisphosphate (PIP2), which is produced by phosphoinositide kinases from phosphatidylinositol. However, the effects of PIP2 on SK channel activity are not known. In the present study, we investigated the mechanism by which hydrolyzable ATP prevented run-down of SK channel activity in excised apical patches of principal cells from rat CCD. Channel run-down was significantly delayed by pretreatment with hydrolyzable Mg-ATP, but ATPγS and AMP-PNP had no effect. Addition of alkaline phosphatase also resulted in loss of channel activity. After run-down, SK channel activity rapidly increased upon addition of PIP2. Exposure of inside-out patches to phosphoinositide kinase inhibitors (LY294002, quercetin or wortmannin) decreased channel activity by 74% in the presence of Mg-ATP. PIP2 added to excised patches reactivated SK channels in the presence of these phosphoinositide kinase inhibitors. The protein kinase A inhibitor, PKI, reduced channel activity by 36% in the presence of Mg-ATP. PIP2 was also shown to modulate the inhibitory effects of extracellular and cytosolic ATP. We conclude that both ATP-dependent formation of PIP2 through membrane-bound phosphoinositide kinases and phosphorylation of SK by PKA play important roles in modulating SK channel activity.

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Cardiac small-conductance calcium-activated potassium channels in health and disease

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-021-02535-0 ◽

2021 ◽

Vol 473 (3) ◽

pp. 477-489 ◽

Cited By ~ 1

Author(s):

Xiao-Dong Zhang ◽

Phung N. Thai ◽

Deborah K. Lieu ◽

Nipavan Chiamvimonvat

Keyword(s):

Ventricular Myocytes ◽

Pulmonary Veins ◽

Differential Sensitivity ◽

Sk Channels ◽

Sk Channel ◽

Ventricular Cells ◽

Intracellular Ca ◽

Life Threatening ◽

Health And Disease ◽

Small Conductance

AbstractSmall-conductance Ca2+-activated K+ (SK, KCa2) channels are encoded by KCNN genes, including KCNN1, 2, and 3. The channels play critical roles in the regulation of cardiac excitability and are gated solely by beat-to-beat changes in intracellular Ca2+. The family of SK channels consists of three members with differential sensitivity to apamin. All three isoforms are expressed in human hearts. Studies over the past two decades have provided evidence to substantiate the pivotal roles of SK channels, not only in healthy heart but also with diseases including atrial fibrillation (AF), ventricular arrhythmia, and heart failure (HF). SK channels are prominently expressed in atrial myocytes and pacemaking cells, compared to ventricular cells. However, the channels are significantly upregulated in ventricular myocytes in HF and pulmonary veins in AF models. Interests in cardiac SK channels are further fueled by recent studies suggesting the possible roles of SK channels in human AF. Therefore, SK channel may represent a novel therapeutic target for atrial arrhythmias. Furthermore, SK channel function is significantly altered by human calmodulin (CaM) mutations, linked to life-threatening arrhythmia syndromes. The current review will summarize recent progress in our understanding of cardiac SK channels and the roles of SK channels in the heart in health and disease.

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Pip2-Channel Interaction as a Critical Element in Regulation of SK Channel Activity

Biophysical Journal ◽

10.1016/j.bpj.2013.11.4127 ◽

2014 ◽

Vol 106 (2) ◽

pp. 749a

Author(s):

Miao Zhang ◽

Meng Cui ◽

Xuan-Yu Meng ◽

Ji-Fang Zhang ◽

Diomedes E. Logothetis

Keyword(s):

Critical Element ◽

Channel Activity ◽

Channel Interaction ◽

Sk Channel

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Abstract 17319: Distinct Effects of Human Calmodulinopathy on the Function of Small Conductance Ca 2+ -Activated K + (SK) Channels

Circulation ◽

10.1161/circ.138.suppl_1.17319 ◽

2018 ◽

Vol 138 (Suppl_1) ◽

Author(s):

Hannah A Ledford ◽

Seojin Park ◽

Duncan Muir ◽

Wen Smith ◽

Ryan L Woltz ◽

...

Keyword(s):

Ion Channels ◽

Intracellular Signaling ◽

Critical Role ◽

Dominant Negative ◽

Polymorphic Ventricular Tachycardia ◽

Dominant Negative Effect ◽

Sk Channels ◽

Channel Function ◽

Sk Channel ◽

Small Conductance

Background: Calmodulin (CaM) plays a critical role in intracellular signaling and regulation of Ca 2+ -dependent ion channels. Mutations in CALM1, CALM2, and CALM3 have recently been linked to cardiac arrhythmias, such as Long QT Syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT), and familial idiopathic ventricular fibrillation (IVF). Small-conductance Ca 2+ - activated K + channels (SK) are voltage-independent channels that are regulated solely from beat-to-beat changes in intracellular calcium. CaM regulates the function of multiple ion channels, including SK channels, although the effect of CaM mutations on these channels is not yet understood. We hypothesize that human CaM mutations linked to sudden cardiac death disrupt SK channel function by distinct mechanisms. Methods and Results: We tested the effects of LQTS (CaM D96V , CaM D130G ), CPVT (CaM N54I , CaM N98S ), and IVF (CaM F90L ) CaM mutants compared to CaM WT on SK channel function. Using whole-cell voltage-clamp recordings, we found that CaM D96V and CaM D130G mutants significantly inhibited apamin-sensitive currents. Similarly, action potential studies in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) also revealed significant knockdown of apamin-sensitive currents. Immunofluorescent confocal microscopy confirmed that this effect was not due to changes in SK channel trafficking. Rather, co-immunoprecipitation studies showed a significant decrease in the association of these CaM mutants with the SK channel. Rosetta molecular modeling was used to identify a conformational change in CaM F90L structure compared to that of CaM WT . Conclusions: We found that CaM D96V and CaM D130G mutants significantly reduced apamin-sensitive currents, through a dominant negative effect on SK channel function. Consistent with our hypothesis, CaM F90L resulted in the least inhibitory effects. The data suggests that specific mutations with phenylalanine to leucine (CaM F90L ) may disrupt the interaction between apo-CaM with CaMBD on the SK2 channel.

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Fragment-based drug design of nature-inspired compounds

Physical Sciences Reviews ◽

10.1515/psr-2018-0110 ◽

2019 ◽

Vol 4 (9) ◽

Cited By ~ 1

Author(s):

Abdulkarim Najjar ◽

Abdurrahman Olğaç ◽

Fidele Ntie-Kang ◽

Wolfgang Sippl

Keyword(s):

Drug Design ◽

Small Molecules ◽

Small Molecule ◽

Binding Pocket ◽

Biological Macromolecules ◽

Drug Candidates ◽

Fragment Screening ◽

Small Molecule Drugs ◽

Lead Generation ◽

Hit Identification

Abstract Natural product (NP)-derived drugs can be extracts, biological macromolecules, or purified small molecule substances. Small molecule drugs can be originally purified from NPs, can represent semisynthetic molecules, natural fragments containing small molecules, or are fully synthetic molecules that mimic natural compounds. New semisynthetic NP-like drugs are entering the pharmaceutical market almost every year and reveal growing interests in the application of fragment-based approaches for NPs. Thus, several NP databases were constructed to be implemented in the fragment-based drug design (FBDD) workflows. FBDD has been established previously as an approach for hit identification and lead generation. Several biophysical and computational methods are used for fragment screening to identify potential hits. Once the fragments within the binding pocket of the protein are identified, they can be grown, linked, or merged to design more active compounds. This work discusses applications of NPs and NP scaffolds to FBDD. Moreover, it briefly reviews NP databases containing fragments and reports on case studies where the approach has been successfully applied for the design of antimalarial and anticancer drug candidates.

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Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer

Nature Communications ◽

10.1038/s41467-019-13616-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 12

Author(s):

James O’Connell ◽

John Porter ◽

Boris Kroeplien ◽

Tim Norman ◽

Stephen Rapecki ◽

...

Keyword(s):

Small Molecules ◽

Small Molecule ◽

Protein Interactions ◽

General Mechanism ◽

Protein Protein Interactions ◽

Biologic Drugs ◽

Small Molecule Drugs ◽

Necrosis Factor

AbstractTumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn’s disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein–protein interactions.

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Pharmacological Modulation of the Gating Properties of Small Conductance Ca2+-Activated K+ Channels Alters the Firing Pattern of Dopamine Neurons In Vivo

Journal of Neurophysiology ◽

10.1152/jn.01126.2009 ◽

2010 ◽

Vol 104 (3) ◽

pp. 1726-1735 ◽

Cited By ~ 21

Author(s):

Kjartan F. Herrik ◽

Palle Christophersen ◽

Paul D. Shepard

Keyword(s):

Firing Rate ◽

Dopamine Neurons ◽

Substantia Nigra Pars Compacta ◽

Pacemaker Activity ◽

Sk Channels ◽

Pharmacological Modulation ◽

Sk Channel ◽

Discharge Patterns ◽

Small Conductance

Dopamine (DA) neurons are autonomous pacemakers that occasionally fire bursts of action potentials, discharge patterns thought to reflect tonic and phasic DA signaling, respectively. Pacemaker activity depends on the concerted and cyclic interplay between intrinsic ion channels with small conductance Ca2+-activated K+ (SK) channels playing an important role. Bursting activity is synaptically initiated but neither the transmitters nor the specific ion conductances involved have been definitively identified. Physiological and pharmacological regulation of SK channel Ca2+ sensitivity has recently been demonstrated and could represent a powerful means of modulating the expression of tonic/phasic signaling in DA neurons in vivo. To test this premise, we characterized the effects of intravenous administration of the novel positive and negative SK channel modulators NS309 and NS8593, respectively, on the spontaneous activity of substantia nigra pars compacta DA neurons in anesthetized C57BL/6 mice. NS309, dose-dependently decreased DA cell firing rate, increased the proportion of regular firing cells, and eventually stopped spontaneous firing. By contrast, systemic administration of the negative SK channel modulator NS8593 increased firing rate and shifted the pattern toward increased irregularity/bursting; an effect similar to local application of the pore blocking peptide apamin. The altered firing patterns resulting from inhibiting SK currents persisted independently of changes in firing rates induced by administration of DA autoreceptor agonists/antagonists. We conclude that pharmacological modulation of SK channel Ca2+-sensitivity represents a powerful mechanism for switching DA neuron firing activity between tonic and phasic signaling modalities in vivo.

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