Channelopathy of Small- and Intermediate-Conductance Ca\u3csup\u3e2+\u3c/sup\u3e-activated K\u3csup\u3e+\u3c/sup\u3e Channels

Small- and intermediate-conductance Ca2+-activated K+ (KCa2.x/KCa3.1 also called SK/IK) channels are gated exclusively by intracellular Ca2+. The Ca2+ binding protein calmodulin confers sub-micromolar Ca2+ sensitivity to the channel-calmodulin complex. The calmodulin C-lobe is constitutively associated with the proximal C-terminus of the channel. Interactions between calmodulin N-lobe and the channel S4-S5 linker are Ca2+-dependent, which subsequently trigger conformational changes in the channel pore and open the gate. KCNN genes encode four subtypes, including KCNN1 for KCa2.1 (SK1), KCNN2 for KCa2.2 (SK2), KCNN3 for KCa2.3 (SK3), and KCNN4 for KCa3.1 (IK). The three KCa2.x channel subtypes are expressed in the central nervous system and the heart. The KCa3.1 subtype is expressed in the erythrocytes and the lymphocytes, among other peripheral tissues. The impact of dysfunctional KCa2.x/KCa3.1 channels on human health has not been well documented. Human loss-of-function KCa2.2 mutations have been linked with neurodevelopmental disorders. Human gain-of-function mutations that increase the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels have been associated with Zimmermann-Laband syndrome and hereditary xerocytosis, respectively. This review article discusses the physiological significance of KCa2.x/KCa3.1 channels, the pathophysiology of the diseases linked with KCa2.x/KCa3.1 mutations, the structure–function relationship of the mutant KCa2.x/KCa3.1 channels, and potential pharmacological therapeutics for the KCa2.x/KCa3.1 channelopathy

Channelopathy of Small- and Intermediate-Conductance Ca\u3csup\u3e2+\u3c/sup\u3e-activated K\u3csup\u3e+\u3c/sup\u3e Channels

Small- and intermediate-conductance Ca2+-activated K+ (KCa2.x/KCa3.1 also called SK/IK) channels are gated exclusively by intracellular Ca2+. The Ca2+ binding protein calmodulin confers sub-micromolar Ca2+ sensitivity to the channel-calmodulin complex. The calmodulin C-lobe is constitutively associated with the proximal C-terminus of the channel. Interactions between calmodulin N-lobe and the channel S4-S5 linker are Ca2+-dependent, which subsequently trigger conformational changes in the channel pore and open the gate. KCNN genes encode four subtypes, including KCNN1 for KCa2.1 (SK1), KCNN2 for KCa2.2 (SK2), KCNN3 for KCa2.3 (SK3), and KCNN4 for KCa3.1 (IK). The three KCa2.x channel subtypes are expressed in the central nervous system and the heart. The KCa3.1 subtype is expressed in the erythrocytes and the lymphocytes, among other peripheral tissues. The impact of dysfunctional KCa2.x/KCa3.1 channels on human health has not been well documented. Human loss-of-function KCa2.2 mutations have been linked with neurodevelopmental disorders. Human gain-of-function mutations that increase the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels have been associated with Zimmermann-Laband syndrome and hereditary xerocytosis, respectively. This review article discusses the physiological significance of KCa2.x/KCa3.1 channels, the pathophysiology of the diseases linked with KCa2.x/KCa3.1 mutations, the structure-function relationship of the mutant KCa2.x/KCa3.1 channels, and potential pharmacological therapeutics for the KCa2.x/KCa3.1 channelopathy

Loss-of-Function K\u3csub\u3eCa\u3c/sub\u3e2.2 Mutations Abolish Channel Activity

Small-conductance Ca2+-activated potassium channels subtype 2 (KCa2.2, also called SK2) are operated exclusively by a Ca2+-calmodulin gating mechanism. Heterozygous genetic mutations of KCa2.2 channels have been associated with autosomal dominant neurodevelopmental disorders including cerebellar ataxia and tremor in humans and rodents. Taking advantage of these pathogenic mutations, we performed structure-function studies of the rat KCa2.2 channel. No measurable current was detected from HEK293 cells heterologously expressing these pathogenic KCa2.2 mutants. When co-expressed with the KCa2.2_WT channel, mutations of the pore-lining amino acid residues (I360M, Y362C, G363S and I389V) and two proline substitutions (L174P and L433P) dominant negatively suppressed and completely abolished the activity of the co-expressed KCa2.2_WT channel. Co-expression of the KCa2.2_I289N and the KCa2.2_WT channels reduced the apparent Ca2+ sensitivity compared with the KCa2.2_WT channel, which was rescued by a KCa2.2 positive modulator

K\u3csub\u3eCa\u3c/sub\u3e2 and K\u3csub\u3eCa\u3c/sub\u3e3.1 Channels in the Airways: A New Therapeutic Target

K+ channels are involved in many critical functions in lung physiology. Recently, the family of Ca2+-activated K+ channels (KCa) has received more attention, and a massive amount of effort has been devoted to developing selective medications targeting these channels. Within the family of KCa channels, three small-conductance Ca2+-activated K+ (KCa2) channel subtypes, together with the intermediate-conductance KCa3.1 channel, are voltage-independent K+ channels, and they mediate Ca2+-induced membrane hyperpolarization. Many KCa2 channel members are involved in crucial roles in physiological and pathological systems throughout the body. In this article, different subtypes of KCa2 and KCa3.1 channels and their functions in respiratory diseases are discussed. Additionally, the pharmacology of the KCa2 and KCa3.1 channels and the link between these channels and respiratory ciliary regulations will be explained in more detail. In the future, specific modulators for small or intermediate Ca2+-activated K+ channels may offer a unique therapeutic opportunity to treat muco-obstructive lung diseases

K\u3csub\u3eCa\u3c/sub\u3e2.2 (KCNN2): A Physiologically and Therapeutically Important Potassium Channel

One group of the K+ ion channels, the small-conductance Ca2+-activated potassium channels (KCa2.x, also known as SK channels family), is widely expressed in neurons as well as the heart, endothelial cells, etc. They are named small-conductance Ca2+-activated potassium channels (SK channels) due to their comparatively low single-channel conductance of about ~10 pS. These channels are insensitive to changes in membrane potential and are activated solely by rises in the intracellular Ca2+. According to the phylogenic research done on the KCa2.x channels family, there are three channels\u27 subtypes: KCa2.1, KCa2.2, and KCa2.3, which are encoded by KCNN1, KCNN2, and KCNN3 genes, respectively. The KCa2.x channels regulate neuronal excitability and responsiveness to synaptic input patterns. KCa2.x channels inhibit excitatory postsynaptic potentials (EPSPs) in neuronal dendrites and contribute to the medium afterhyperpolarization (mAHP) that follows the action potential bursts. Multiple brain regions, including the hippocampus, express the KCa2.2 channel encoded by the KCNN2 gene on chromosome 5. Of particular interest, rat cerebellar Purkinje cells express KCa2.2 channels, which are crucial for various cellular processes during development and maturation. Patients with a loss-of-function of KCNN2 mutations typically exhibit extrapyramidal symptoms, cerebellar ataxia, motor and language developmental delays, and intellectual disabilities. Studies have revealed that autosomal dominant neurodevelopmental movement disorders resembling rodent symptoms are caused by heterozygous loss-of-function mutations, which are most likely to induce KCNN2 haploinsufficiency. The KCa2.2 channel is a promising drug target for spinocerebellar ataxias (SCAs). SCAs exhibit the dysregulation of firing in cerebellar Purkinje cells which is one of the first signs of pathology. Thus, selective KCa2.2 modulators are promising potential therapeutics for SCAs

Channelopathy-Causing Mutations in the S\u3csub\u3e45\u3c/sub\u3eA/S\u3csub\u3e45\u3c/sub\u3eB and HA/HB Helices of K\u3csub\u3eCa\u3c/sub\u3e2.3 and K\u3csub\u3eCa\u3c/sub\u3e3.1 Channels Alter Their Apparent Ca\u3csup\u3e2+\u3c/sup\u3e Sensitivity

Small- and intermediate-conductance Ca2+-activated potassium (KCa2.x and KCa3.1, also called SK and IK) channels are activated exclusively by a Ca2+-calmodulin gating mechanism. Wild-type KCa2.3 channels have a Ca2+ EC50 value of ∼0.3 μM, while the apparent Ca2+ sensitivity of wild-type KCa3.1 channels is ∼0.27 μM. Heterozygous genetic mutations of KCa2.3 channels have been associated with Zimmermann-Laband syndrome and idiopathic noncirrhotic portal hypertension, while KCa3.1 channel mutations were reported in hereditary xerocytosis patients. KCa2.3_S436C and KCa2.3_V450L channels with mutations in the S45A/S45B helices exhibited hypersensitivity to Ca2+. The corresponding mutations in KCa3.1 channels also elevated the apparent Ca2+ sensitivity. KCa3.1_S314P, KCa3.1_A322V and KCa3.1_R352H channels with mutations in the HA/HB helices are hypersensitive to Ca2+, whereas KCa2.3 channels with the equivalent mutations are not. The different effects of the equivalent mutations in the HA/HB helices on the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels may imply distinct modulation of the two channel subtypes by the HA/HB helices. AP14145 reduced the apparent Ca2+ sensitivity of the hypersensitive mutant KCa2.3 channels, suggesting the potential therapeutic usefulness of negative gating modulators

Subtype-selective Positive Modulation of K\u3csub\u3eCa\u3c/sub\u3e 2 Channels Depends on the HA/HB Helices

Background and Purpose In the activated state of small-conductance Ca2+-activated potassium (KCa 2) channels, calmodulin interacts with the HA/HB helices and the S4-S5 linker. CyPPA potentiates KCa 2.2a and KCa 2.3 channel activity but not the KCa 2.1 and KCa 3.1 subtypes. Experimental Approach Site-directed mutagenesis, patch-clamp recordings and in silico modeling were utilized to explore the structural determinants for the subtype-selective modulation of KCa 2 channels by CyPPA. Key Results Mutating residues in the HA (V420) and HB (K467) helices of KCa 2.2a channels to their equivalent residues in KCa 3.1 channels diminished the potency of CyPPA. CyPPA elicited prominent responses on mutant KCa 3.1 channels with an arginine residue in the HB helix substituted for its equivalent lysine residue in the KCa 2.2a channels (R355K). KCa 2.1 channels harboring a three-amino-acid insertion upstream of the cognate R438 residues in the HB helix showed no response to CyPPA, whereas the deletion mutant (KCa 2.1_ΔA434/Q435/K436) became sensitive to CyPPA. In molecular dynamics simulations, CyPPA docked between calmodulin C-lobe and the HA/HB helices widens the cytoplasmic gate of KCa 2.2a channels. Conclusion and Implications Selectivity of CyPPA among KCa 2 and KCa 3.1 channel subtypes relies on the HA/HB helices

Subtype-Selective Positive Modulation of K\u3csub\u3eCa\u3c/sub\u3e2.3 Channels Increases Cilia Length

Small-conductance Ca2+-activated potassium (KCa2.x) channels are gated exclusively by intracellular Ca2+. The activation of KCa2.3 channels induces hyperpolarization, which augments Ca2+ signaling in endothelial cells. Cilia are specialized Ca2+ signaling compartments. Here, we identified compound 4 that potentiates human KCa2.3 channels selectively. The subtype selectivity of compound 4 for human KCa2.3 over rat KCa2.2a channels relies on an isoleucine residue in the HA/HB helices. Positive modulation of KCa2.3 channels by compound 4 increased flow-induced Ca2+ signaling and cilia length, while negative modulation by AP14145 reduced flow-induced Ca2+ signaling and cilia length. These findings were corroborated by the increased cilia length due to the expression of Ca2+-hypersensitive KCa2.3_G351D mutant channels and the reduced cilia length resulting from the expression of Ca2+-hyposensitive KCa2.3_I438N channels. Collectively, we were able to associate functions of KCa2.3 channels and cilia, two crucial components in the flow-induced Ca2+ signaling of endothelial cells, with potential implications in vasodilation and ciliopathic hypertension

Pharmacological and Pharmacokinetic Studies of a K\u3csub\u3eCa\u3c/sub\u3e2.2 Positive Allosteric Modulator

Small-conductance Ca2+-activated potassium channels (KCa2.x) family is widely expressed in neurons, the heart, and endothelial cells. KCa2.x channels are named small conductance Ca2+-activated potassium channels due to their comparatively low single-channel conductance and are activated solely by rises in intracellular Ca2+. The family has three subtypes: KCa2.1, KCa2.2, and KCa2.3, encoded by KCNN1, KCNN2, and KCNN3 genes, respectively. KCa2.x channels regulate neuronal excitability and responsiveness to synaptic input patterns. Small-conductance Ca2+-activated potassium channels subtype 2 (KCa2.2, also called SK2) is a promising drug target for spinocerebellar ataxias (SCAs), genetic disorders with no available treatment. Heterozygous genetic mutations of KCa2.2 channels have been associated with autosomal dominant neurodevelopmental disorders, including cerebellar ataxia and tremor in humans and rodents. The structure-function studies of the rat KCa2.2 channel using seven pathogenic mutations (I289N, I360M, Y362C, G363S, I389V, L174P, and L433P) associated with these disorders were performed to investigate the insight of these disorders related to KCa2.2. The dominant mutations negatively suppressed and completely abolished the activity of the co-expressed KCa2.2_WT channel, suggesting that these mutations may be causative in neurodevelopmental disorders. Additionally, the co-expression of the KCa2.2_I289N and the KCa2.2_WT channels reduced the apparent Ca2+ sensitivity compared with the KCa2.2_WT channel rescued by a KCa2.2 positive modulator. A positive allosteric modulator of KCa2.2/KCa2.3 channels (compound 2q) has been developed, and a method for quantitating it in mouse plasma has been validated using FDA guidelines. The developed assay is suitable for preclinical pharmacokinetic-pharmacodynamic studies of 2q as a potential drug candidate for ataxias

Channelopathy-causing mutations in the S45A/S45B and HA/HB helices of KCa2.3 and KCa3.1 channels alter their apparent Ca2+ sensitivity

Small- and intermediate-conductance Ca2+-activated potassium (KCa2.x and KCa3.1, also called SK and IK) channels are activated exclusively by a Ca2+-calmodulin gating mechanism. Wild-type KCa2.3 channels have a Ca2+ EC50 value of ∼0.3 μM, while the apparent Ca2+ sensitivity of wild-type KCa3.1 channels is ∼0.27 μM. Heterozygous genetic mutations of KCa2.3 channels have been associated with Zimmermann-Laband syndrome and idiopathic noncirrhotic portal hypertension, while KCa3.1 channel mutations were reported in hereditary xerocytosis patients. KCa2.3_S436C and KCa2.3_V450L channels with mutations in the S45A/S45B helices exhibited hypersensitivity to Ca2+. The corresponding mutations in KCa3.1 channels also elevated the apparent Ca2+ sensitivity. KCa3.1_S314P, KCa3.1_A322V and KCa3.1_R352H channels with mutations in the HA/HB helices are hypersensitive to Ca2+, whereas KCa2.3 channels with the equivalent mutations are not. The different effects of the equivalent mutations in the HA/HB helices on the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels may imply distinct modulation of the two channel subtypes by the HA/HB helices. AP14145 reduced the apparent Ca2+ sensitivity of the hypersensitive mutant KCa2.3 channels, suggesting the potential therapeutic usefulness of negative gating modulators