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. Author manuscript; available in PMC: 2023 Feb 1.
Published in final edited form as: Br J Pharmacol. 2021 Oct 1;179(3):460–472. doi: 10.1111/bph.15676

Subtype-selective positive modulation of KCa 2 channels depends on the HA/HB helices

Young-Woo Nam 1,*, Meng Cui 2,*, Naglaa Salem El-Sayed 1, Razan Orfali 1, Misa Nguyen 1, Grace Yang 1, Mohammad Asikur Rahman 1, Judy Lee 1, Miao Zhang 1
PMCID: PMC8799485  NIHMSID: NIHMS1738338  PMID: 34458981

Abstract

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.

Keywords: ion channel, patch-clamp, CyPPA, small-conductance Ca2+-activated potassium channel

1 |. INTRODUCTION

KCa 2 and KCa 3.1 channels are a unique group of potassium ion channels that are activated exclusively by intracellular Ca2+. The Ca2+-binding protein calmodulin (CaM) is constitutively bound to these channels and serves as their Ca2+ sensor (Adelman, Maylie, & Sah, 2012). There are four mammalian genes identified in the KCNN family, including KCNN1 for KCa 2.1 (SK1), KCNN2 for KCa 2.2 (SK2), KCNN3 for KCa 2.3 (SK3) and KCNN4 for KCa 3.1 (SK4 or IK) channels, respectively. The three KCa 2 channel subtypes KCa 2.1, KCa 2.2 and KCa 2.3 are expressed in neurons. KCa 3.1 channels are predominantly distributed in peripheral tissues, including secretory epithelia and erythrocytes, T-cells, macrophages and microglia (Brown, Shim, Christophersen, & Wulff, 2020).

In neurons, activation of KCa 2 channels leads to reduced neuronal activity through their involvement in the medium afterhyperpolarization (mAHP) (Pedarzani et al., 2001; Stocker, 2004). Neuronal KCa 2 channels have been proposed as novel drug targets for spinocerebellar ataxias (SCAs) and other movement disorders (Kasumu, Hougaard, et al., 2012; Shakkottai et al., 2011; Walter, Alvina, Womack, Chevez, & Khodakhah, 2006). Disruptions of regular pacemaking activity of Purkinje cells have been identified in studies with mouse models of SCA3 (Shakkottai et al., 2011) and SCA2 (Kasumu, Liang, Egorova, Vorontsova, & Bezprozvanny, 2012). KCa 2 channels emerged as one of the principal ion channels involved in Purkinje cells pacemaking (Womack & Khodakhah, 2003). Among the three KCa 2 channel subtypes, the KCa 2.2 channel subtype is the predominant subtype expressed in Purkinje cells (Cingolani, Gymnopoulos, Boccaccio, Stocker, & Pedarzani, 2002; Hosy, Piochon, Teuling, Rinaldo, & Hansel, 2011; Sailer, Kaufmann, Marksteiner, & Knaus, 2004). An isoleucine-to-asparagine (I289N) mutation was reported to cause diminished KCa 2.2 channel current and tremor in the tremor dominant Kyoto (Trdk) rats (Kuramoto et al., 2017). A similar phenotype was observed in human caused by a glycine-to-glutamic acid (G371E) mutation of the KCNN2 gene (Balint et al., 2020). Indeed, loss-of-function mutations in the KCNN2 gene may lead to neurodevelopmental movement disorders including cerebellar ataxia (Mochel et al., 2020). In mouse models of SCAs, positive KCa 2 channel modulators like Chlorzoxazone (Egorova, Zakharova, Vlasova, & Bezprozvanny, 2016), SKA31 (Shakkottai et al., 2011) and NS13001 (Kasumu, Hougaard, et al., 2012) exert beneficial effects through potentiating the KCa 2.2 channel subtype expressed in cerebellar Purkinje cells.

It is critical to identify subtype-selective KCa 2 channel modulators in order to minimize off-target side effects. The prototypical subtype-selective positive modulator that enhances KCa 2.2 and KCa 2.3 channel activity but not the KCa 2.1 and KCa 3.1 channel activity is CyPPA (Hougaard et al., 2007). CyPPA and its analog NS13001 showed promise in alleviating symptoms in animal models of ataxia (Kasumu, Hougaard, et al., 2012).

CyPPA exerts its effects on channel activity through influencing the apparent Ca2+ sensitivity (Hougaard et al., 2007). But how CyPPA interacts with the KCa 2.2 channels remains unclear. KCa 2 and KCa 3.1 channels are tetramers with each subunit having six transmembrane α-helical domains denoted S1–S6. Limited by the structural information available, the search for the binding site of the KCa 2 channel positive modulators until recently was focused on a proximal C-terminal region named the CaM-binding domain (CaMBD) that interacts with CaM (Cho et al., 2018; Zhang, Pascal, Schumann, Armen, & Zhang, 2012; Zhang, Pascal, & Zhang, 2013). Crystallography studies of the CaM/CaMBD complex revealed a putative binding site of a CyPPA analog between the N-lobe of CaM and the CaMBD (Cho et al., 2018). A recent cryo-EM study of the KCa 3.1 (SK4) channel subtype demonstrated that the N-lobe of CaM may interact with the linker between S4 and S5 transmembrane domains (S4-S5 linker), instead of the CaMBD (Lee & MacKinnon, 2018). Since the S4-S5 linker was not included in the crystallography study of the CaM/CaMBD complex (Cho et al., 2018), the N-lobe of CaM formed binding interface with the CaMBD instead. As such, the reported binding site of the CyPPA analog (Cho et al., 2018) between the N-lobe of CaM and the CaMBD may not exist in the full-length KCa 2 channel, because the N-lobe of CaM interacts with the S4-S5 linker in the full-length structure.

Utilizing this cryo-EM structure of human KCa 3.1 channels (PDB code: 6CNN) as a template, we generated a homology model of the rat KCa 2.2a channels as previously described (Nam et al., 2018). The CaMBD consists of two a-helices, HA and HB, while the S4-S5 linker includes two α-helices, S45A and S45B. The HA and HB helices from one channel subunit, the S4-S5 linker from a neighboring channel subunit and CaM closely interact with each other. Here, we report that the subtype-selective potentiation of KCa 2.2a channels by CyPPA depends primarily on the HA/HB helices. Mutating an arginine residue on the HB helix of KCa 3.1 channels to its corresponding lysine residue in KCa 2.2a channels (R355K) rendered the channel’s sensitivity to CyPPA. KCa 2.1 channels with a three amino acid (AQK) insertion in the HB helix showed no response to CyPPA, while the deletion mutant (ΔA434/Q435/K436) gained sensitivity to CyPPA.

2 |. METHODS

2.1 |. Electrophysiology

The effect of mutations on the sensitivity of KCa 2 and KCa 3.1 channels to Ca2+ and CyPPA was investigated as previously described (Nam et al., 2018; Nam et al., 2020). Briefly, mutations were introduced to the rat KCa 2.2a, human KCa 2.1 or human KCa 3.1 channels using QuickChange II site-directed mutagenesis kit (Agilent, Santa Clara, CA, USA). The previously reported chimeric channel cDNA constructs swapping the S4-S5 linkers between KCa 2.2a and KCa 3.1 channels were generated through gene synthesis (Genscript, Piscataway, NJ, USA) (Nam et al., 2020). Both the channel cDNAs and CaM cDNAs were constructed in pcDNA3.1+. The mutant channel cDNAs, along with CaM and GFP (pEGFP-C2), at a ratio of 7:4:2 (ORF ratios), were transfected into HEK293 cells (CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045) by the calcium–phosphate method. HEK293 is an immortalised epithelial cell line with an origin of female Homo sapiens. HEK293 is well established for electrophysiology studies of heterologously expressed potassium channels, because of its high transfection efficiency, faithful translation and processing of channel proteins, and low expression of native potassium channels. This study of channel proteins on excised membrane patches heterologously expressed by immortalised cells is not sex specific. The experimental results should apply to both sexes, because the direct pharmacological modulation of heterologously expressed potassium channels is not sex specific. KCa currents were recorded 1–2 days after transfection, with an Axon200B amplifier (Molecular Devices, San Jose, CA, USA) at room temperature.

pClamp 10.5 (Molecular Devices, San Jose, CA, USA, RRID:SCR_011323) was used for data acquisition and analysis. The resistance of the patch electrodes ranged from 3–5 MΩ. The pipette solution contained (in mM): 140 KCl, 10 Hepes (pH 7.4), 1 MgSO4. The bath solution containing (in mM): 140 KCl, 10 Hepes (pH 7.2), 1 EGTA, 0.1 Dibromo-BAPTA, and 1 HEDTA was mixed with Ca2+ to obtain the desired free Ca2+ concentrations, calculated using the software by Chris Patton of Stanford University (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/webmaxc/webmaxcS.htm). The Ca2+ concentrations were verified using a Ca2+ calibration buffer kit (Thermo Fisher Scientific). Briefly, a standard curve was generated using the Ca2+ buffers from the kit and a fluorescence Ca2+ indicator. Then the Ca2+ concentrations of the bath solution were determined through interpolation on the standard curve.

High resistance seals (> 1 GΩ) were formed before inside-out patches were obtained. The seal resistance of inside-out patches was > 1 GΩ, when the intracellular face was initially exposed to a zero-Ca2+ bath solution. The intracellular face was subsequently exposed to bath solutions with a series of Ca2+ concentrations to measure Ca2+ sensitivity. Currents were recorded by repetitive 1-s-voltage ramps from − 100 mV to + 100 mV from a holding potential of 0 mV. The currents were filtered at 2 kHz and digitized at a sampling frequency of 10 kHz. One minute after switching of bath solutions, ten sweeps with a 1-s interval were recorded. The integrity of the patch was examined by switching the bath solution back to the zero-Ca2+ buffer. Data from patches, which maintained the seal resistance (> 1 GΩ) after solution changes, were used for further analysis. To construct the concentration-dependent activation of channel activities, the current amplitudes at − 90 mV in response to various concentrations of Ca2+ were normalised to that obtained at maximal concentration of Ca2+. The normalised currents were plotted as a function of the concentrations of Ca2+. EC50 values and Hill coefficients were determined by fitting the data points to a standard concentration–response curve (Y = 100/(1 + (X/EC50)^ − Hill)).

To measure the sensitivity to CyPPA (Alomone Labs, Jerusalem, Israel), the intracellular face was exposed to bath solutions with 0.15 μM Ca2+ unless otherwise indicated. One minute after switching of bath solutions, ten sweeps with a 1-s interval were recorded at a series of concentrations of CyPPA in the presence of 0.15 μM Ca2+ unless otherwise indicated. The maximal KCa 2 and KCa 3.1 current in response to 10 μM Ca2+ was then recorded. To construct the concentration-dependent potentiation of channel activities by CyPPA, the current amplitudes at − 90 mV in response to various concentrations of CyPPA were normalised to that obtained at maximal concentration of CyPPA. The normalised currents were plotted as a function of the concentrations of CyPPA. EC50 values and Hill coefficients were determined by fitting the data points to a standard concentration–response curve (Y = 100/(1 + (X/EC50)^ − Hill)). To assess the efficacy of CyPPA, the current amplitudes obtained at maximal concentration of CyPPA were normalised to the maximal KCa 2 and KCa 3.1 current in response to 10 μM Ca2+.

2.2 |. MD simulations

The homology model of the rat KCa 2.2a channel was described in our previous report (Nam et al., 2018). We built homology model for the KCa 2.1 channel based on a cryo-EM structure of KCa 3.1 channel (PDB Code: 6CNN). Sequence alignment among KCa 2.1, KCa 2.2 and KCa 3.1 channels was generated by Clustal Omega server (https://www.ebi.ac.uk/Tools/msa/clustalo/, RRID:SCR001591). The sequence identity between KCa 2.1 and KCa 3.1 is 43.3%, which makes the KCa 3.1 Cryo-EM structure an excellent structural template to generate homology models for the KCa 2.1 channels. We used the MODELLER program (RRID: SCR008395) (Sali & Blundell, 1993) to generate 10 initial homology models for KCa 2.1 channel based on the KCa 3.1 structural template, and selected the one with the best internal DOPE score from the program.

KCa 2.2a channel model structure was prepared by the Protein Preparation Wizard module of Maestro (2019–3) program (Schrödinger, Inc. New York, NY). The binding site of CyPPA in the KCa 2.2a channel was defined by residue V420 and K467, based on mutagenesis results. A grid box of 30Å × 30Å × 30Å was generated using Receptor Grid generation module. The default parameters were used for the Grid generation. Flexible ligand dockings were performed using standard precision (SP) module in Glide program (Friesner et al., 2004). The docked pose with the lowest SP score was selected, and used as a reference ligand for induced-fit-docking (IFD) simulations (Sherman, Day, Jacobson, Friesner, & Farid, 2006). The default parameters were used for IFD simulations. The residues within 5Å of ligand poses were selected for side chain optimization by prime refinement. The SP score was used for ranking of the ligand poses, and top five poses of docked ligand were saved for visual inspection and selection. The pose of docked CyPPA with the lowest docking SP score was selected for MD simulations.

For MD simulations, the KCa 2.2a_apo, KCa 2.2a_1_CyPPA, KCa 2.2a_2_CyPPA, KCa 2.2a_3_CyPPA and KCa 2.2a_4_CyPPA structures were immersed in an explicit lipid bilayer of POPC, POPE, POPS and cholesterol with molecular ratio of 25:5:5:1 (Leal-Pinto, London, Knorr, & Abramson, 1995), and a water box in 168.1Å × 165.6Å × 140.0Å dimension by using the CHARMM-GUI Membrane Builder webserver (http://www.charmm-gui.org/?doc=input/membrane). Ca2+ ions were added to the EF hands of CaM according to the cryo-EM structure of KCa 3.1 channels (PDB code: 6CNN). 150mM KCl and extra neutralizing counter ions were added into the systems. The total atoms of the systems were 293,068, 293,141, 293,185, 293,229 and 293,273 for the KCa 2.2a_apo, KCa 2.2a_1_CyPPA, KCa 2.2a_2_CyPPA, KCa 2.2a_3_CyPPA and KCa 2.2a_4_CyPPA, respectively. The Antechamber module of AmberTools was used to generate the parameters for CyPPA using general AMBER force field (GAFF). The partial charges for the CyPPA were calculated using restrained electrostatic potential (RESP) charge-fitting scheme by ab initio quantum chemistry at the HF/6–31G* level (GAUSSIAN 16 program) (Breneman & Wiberg, 1990). The PMEMD.CUDA program in AMBER16 was used to conduct the MD simulations. The MD simulations were performed with periodic boundary conditions to produce isothermal-isobaric ensembles. Long range electrostatics were calculated using the particle mesh Ewald (PME) method (Darden, 1993) with a 10 Å cutoff. Prior to production runs, energy minimization of the system was carried out. Subsequently, the system was heated from 0 K to 303K using Langevin dynamics with the collision frequency of 1 ps−1. During the heating, the KCa 2.2a channel was position-restrained using an initial constant force of 500 kcal/mol/Å2, and weakened to 10 kcal/mol/Å2, allowing lipid and water molecules to move freely. Then, the system went through 5 ns of relaxation. Finally, a total of 200 or 500 ns production MD simulation was conducted, and coordinates were saved every 100 ps for analysis. In total, 5,000 snapshot structures were collected during the MD simulations. The minimum distances between atoms in the trajectories were analysed using the built-in utility of GROMACS program (Hess, Kutzner, van der Spoel, & Lindahl, 2008).

2.3 |. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). Patch-clamp recordings were analysed using Clampfit 10.5 (Molecular Devices LLC, San Jose, CA, USA, RRID:SCR_011323) and concentration-response curves were analysed in GraphPad Prism 9.0.2 (GraphPad Software Inc., La Jolla, CA, USA, RRID: SCR_002798). The responses to various concentrations of CyPPA or Ca2+ were normalised to that obtained at maximal concentration of CyPPA or Ca2+, to control for unwanted sources of variation including the size of the inside-out patches and/or the expression level of channels. If normalisation generated values with no variance, the data point was not subjected to parametric statistical analysis. Concentration-response curves were acquired from multiple patches for each data set. Each curve was fitted individually, which yielded the EC50 value for that curve. EC50 values are shown as mean ± SEM obtained from multiple transfections and the number of separate transfections is indicated by n.

We did not perform randomization because we did not have animals or human subjects in our study. The cell transfection with channel cDNAs, the patch-clamp recordings and data analysis were performed by three researchers in a blinded fashion. Data with a maximal current in response to 10 μM Ca2+ out of the range between 100 and 4000 pA were excluded from the data set, and this caused the variable group sizes. All electrophysiology experimental results included at least five patches.

The Student’s t-test was used for data comparison if there were only two groups. One-way ANOVA and Tukey’s post hoc tests were used for data comparison of three or more groups. Post hoc tests were carried out only if F was significant and there was no variance in homogeneity. P values < 0.05 were considered as statistically significant.

2.4 |. Materials

CyPPA was purchased from Alomone Labs, Jerusalem, Israel. CyPPA was dissolved in DMSO to make stock solution of 100 mM. The stock solution was then diluted in bath solution to the final concentrations for patch-clamp recordings.

2.5 |. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019).

3 |. RESULTS

3.1 |. Mutagenesis of the S4-S5 linker

CyPPA selectively potentiates the activity of KCa 2.2a and KCa 2.3 channels, but is inactive on the KCa 2.1 and KCa 3.1 channel subtypes. However, it was unclear how CyPPA achieves such subtype-selectivity towards KCa 2.2a and KCa 2.3 channels. To test whether differences in the amino acid sequence of the S4-S5 linker (Figure 1A) contribute to the selectivity of CyPPA, we utilized two chimeric channel cDNAs with the S4-S5 linkers swapped between the KCa 2.2a and KCa 3.1 channel subtypes, described in our recent report (Nam et al., 2020). The KCa 3.1_S4-S5-KCa 2.2 chimera is composed of the transmembrane domains, N- and C-termini of KCa 3.1 channels, together with the S4-S5 linker of KCa 2.2a channels. In contrast, replacing the S4-S5 linker of the KCa 2.2a channel with that of the KCa 3.1 channels generated the KCa 2.2a_S4-S5-KCa 3.1 chimera. We first examined the effect of these mutations on the Ca2+-dependent channel activation using inside-out patch-clamp recordings on the mutant channels heterologously expressed in HEK293 cells (Figure S1A, B). Neither the KCa 3.1_S4-S5-KCa 2.2 nor the KCa 2.2a_S4-S5-KCa 3.1 channel changed the apparent Ca2+ sensitivity compared with their respective WT channels (Figure S1C, D). Positive modulators of KCa 2 channels like CyPPA require minimal concentration of Ca2+ to be effective (Hougaard et al., 2007). Therefore, we measured the concentration-dependent responses of the WT and chimeric channels to CyPPA in the presence of 0.15 μM Ca2+. The KCa 3.1_S4-S5-KCa 2.2 chimeric channel exhibited a very limited response to CyPPA, similar to the WT KCa 3.1 channels (Figure 1B). Consistently, the responses of the KCa 2.2a_S4-S5-KCa 3.1 chimeric channel to CyPPA did not differ from the responses of the WT KCa 2.2a channel (Figure 1B).

FIGURE 1. The sensitivity of KCa 2.2a channels to CyPPA is not affected by the S4-S5 linker.

FIGURE 1

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(A) Amino acid sequence alignment of rat KCa 2.2a [GenBank: NP_062187.1] and human KCa 3.1 [GenBank: NP_002241.1] channels at the S4-S5 linker. The S45A and S45B helices are highlighted in yellow. Mutated residues are shown in bold fonts. (B) Concentration-dependent potentiation by CyPPA of the WT, chimeric and mutant KCa 3.1 and KCa 2.2a channels. The responses were normalised by the maximal currents induced by 10 μM Ca2+. (C) Emax to CyPPA of the WT, chimeric and mutant KCa 3.1 and KCa 2.2a channels. No statistical significance between the chimeric and mutant channels, as compared to their respective WT channel subtype.

To evaluate the efficacy (Emax) of CyPPA on the WT and mutant KCa 2.2a channels, the current amplitudes at − 90 mV in response to the maximal concentration of CyPPA were normalised to that obtained at 10 μM Ca2+ (I / Imax (%), Figure 1B). The responses induced by 10 μM Ca2+ are considered as the maximal currents of the KCa 2.2a channels and the efficacy of CyPPA was reported to be ~71% of that maximal current on the human KCa 2.2a channels (Hougaard et al., 2007). In our hands, CyPPA exhibited an Emax of 81.17 ± 4.30 % on the WT rat KCa 2.2a channels, which is comparable to its Emax of 76.82 ± 3.29 % on the KCa 2.2a_S4-S5-KCa 3.1 chimeric channel (Figure 1C). Consistently, CyPPA barely elicited any response on WT KCa 3.1 channels with an Emax of 14.98 ± 4.19 %, similar to its Emax of 22.13 ± 5.70 % on the KCa 3.1_S4-S5-KCa 2.2 chimeric channel (Figure 1C).

Even though swapping the S4-S5 linkers between the KCa 2.2a and KCa 3.1 channel subtypes did not change their responses towards CyPPA, it does not rule out the potential interactions of CyPPA with the S4-S5 linker. To test this possibility, we introduced alanine substitutions of the S288 and L292 residues, and mutated the A291 residue to phenylalanine in KCa 2.2a channels. Both KCa 2.2a_S288A_L292A and KCa 2.2a_A291F mutations affected the Ca2+ sensitivity, with reduced EC50 values of 0.99 ± 0.092 μM and 0.64 ± 0.072 μM for Ca2+, respectively (Figure S1D). As a result, the potentiation by CyPPA of the KCa 2.2a_S288A_L292A mutant channels were recorded in the presence of 0.5 μM Ca2+, while the responses to CyPPA of the KCa 2.2a_A291F mutant channels were recorded in the presence of 0.3 μM Ca2+. To evaluate the potency (EC50) of CyPPA on the WT and mutant KCa 2.2a channels, the current amplitudes at − 90 mV in response to various concentrations of CyPPA were normalised to that obtained at maximal concentration of CyPPA. Neither KCa 2.2a_S288A_L292A nor KCa 2.2a_A291F mutation changed the channels’ sensitivity to CyPPA (Figure S1E), with EC50 values of 4.89 ± 1.02 μM and 4.87 ± 0.53 μM, as compared to the EC50 value of 7.05 ± 0.80 μM from the WT channels (Figure S1F). In addition, these mutations did not change the Emax of CyPPA on the KCa 2.2a channel (Figure 1C). Collectively, the S4-S5 linker seemed unlikely to be the major structural determinant for the subtype-selectivity of CyPPA.

3.2 |. Mutagenesis of the proximal C-terminus of KCa 2.2a channels

Next, we aligned the amino acid sequences of the KCa 2.2a and KCa 3.1 channel subtypes around the CaMBD in the proximal C-terminus (Figure 2A). The CaMBD forms two a-helices, HA and HB. The amino acid sequences of the two subtypes at the HA helix show ~68% (25 out of 37 residues) identity, while they are ~47% (14 out of 30 residues) identical in the HB helix. We substituted residues in the KCa 2.2a channel for their equivalent residues in the KCa 3.1 channel. There is no statistically significant difference between apparent Ca2+ sensitivity of these mutant channels with that of the WT KCa 2.2a channel (EC50 = 0.31 ± 0.017 μM) (Figure S2A, B). As such, we measured the concentration-dependent responses of these WT and mutant channels towards CyPPA in the presence of 0.15 μM Ca2+ (Figure S2C, D).

FIGURE 2. Mutations in the HA and HB helices change sensitivity of KCa 2.2a channels to CyPPA.

FIGURE 2

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(A) Amino acid sequence alignment of human KCa 3.1 [GenBank: NP_002241.1] and rat KCa 2.2a [GenBank: NP_062187.1] channels at the proximal channel C-terminus. HA and HB helices are highlighted in green. Mutated residues are shown in bold fonts. V420 and K467 in KCa 2.2a channels and their equivalent residues in KCa 3.1 channels are shown in red font. (B) Potentiation by CyPPA of the WT and mutant KCa 2.2a channels carrying double or triple mutations in the proximal C-terminus. (C) EC50 values for potentiation by CyPPA of the WT and mutant channels. * P < 0.05 compared with WT. No asterisk means no statistical significance compared with WT.

CyPPA concentration-dependently potentiated the activity of the WT and mutant KCa 2.2a channels (Figure 2B). Compared with the WT KCa 2.2a channel (EC50 = 7.05 ± 0.80 μM), the KCa 2.2a_R419E_V420M_N422E and KCa 2.2a_K467R_M468L mutations effectively reduced the sensitivity of KCa 2.2a channels to CyPPA (Figure 2C). EC50 values for CyPPA of these two mutant channels were 19.95 ± 1.84 μM and 14.75 ± 1.05 μM, respectively. Other mutations tested did not affect the sensitivity of KCa 2.2a channel to CyPPA significantly (Figure 2C). None of the mutations tested changed the Emax of CyPPA on the KCa 2.2a channel significantly (Figure S2E, F).

3.3 |. CyPPA in the structure of KCa 2.2a channels

Using a cryo-EM structure of the human KCa 3.1 channel published in 2018 (Lee & MacKinnon, 2018) as template, we generated a homology model of the rat KCa 2.2a channel (Figure 3A) as described previously (Nam et al., 2018). The CaMBD at the proximal C-terminus of the KCa 2.2a channel model appears to form two helices named as HA and HB (Figure 3A, B). Both the HA and HB helices (in green color) have substantial interactions with the CaM C-lobe (in grey color) (Figure 3B).

FIGURE 3. CyPPA is docked into a putative binding site between the HA/HB helices and CaM C-lobe in KCa 2.2a channels.

FIGURE 3

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(A) A homology model of the rat KCa 2.2a channel was generated using the human KCa 3.1 channel cryo-EM structure with Ca2+. (B) CyPPA (salmon) is docked into the binding interface between the HA/HB helices (green) and CaM C-lobe (grey). (C) Concentration-dependent potentiation by CyPPA of the WT and mutant KCa 2.2a channels carrying single mutations in the HA/HB helices. (D) EC50 values for potentiation by CyPPA of the WT and mutant channels. * P < 0.05 compared with WT. No asterisk means no statistical significance compared with WT.

The R419E_V420M_N422E triple mutations and the K467R_M468L double mutations dampened the sensitivity of KCa 2.2a channels to CyPPA (Figure 2B, C). We first examined the impact of each of those residues on the channels’ apparent Ca2+ sensitivity (Figure S3A), before testing CyPPA on the mutant channels. None of these single mutations tested changed the apparent Ca2+ sensitivity of the KCa 2.2a channels (Figure S3B). KCa 2.2a_R419E, KCa 2.2a_N422E and KCa 2.2a_M468L mutations did not affect the potency of CyPPA, with EC50 values of 5.67 ± 0.66 μM, 10.94 ± 0.66 μM and 6.14 ± 0.78 μM, respectively. In contrast, KCa 2.2a_V420M and KCa 2.2a_K467R reduced the channels’ sensitivity to CyPPA significantly, with EC50 values of 17.94 ± 2.04 μM and 18.98 ± 1.79 μM (Figure 3C, D), respectively. None of the single mutations tested changed the Emax of CyPPA on the KCa 2.2a channel significantly (Figure S3C, D).

Next, we took a close look at the two residues, V420 and K467, that affected the potency of CyPPA in the KCa 2.2a channel model (Figure 3B). The V420 residue sits in the HA helix with its side chain approaching towards the K467 residue in the almost parallel HB helix. The two γ-carbon atoms of V420 are within a 5 Å radius from the γ-carbon atom of K467. We then docked the CyPPA molecule (in salmon color) into the region around the two residues V420 and K467. The ζ-nitrogen atom of K421 is 4.4 Å from a nitrogen atom in the pyrimidine moiety of CyPPA, and 4.6 Å from a nitrogen in the pyrazole moiety of CyPPA, implying potential hydrogen bonds. Indeed, the KCa 2.2a_K421A mutation caused a drastic right shift of concentration-response curves of the channels to CyPPA (Figure 3C), with a significantly larger EC50 value of 20.79 ± 2.18 μM (Figure 3D).

3.4 |. Mutagenesis of the proximal C-terminus of KCa 3.1 channels to render CyPPA sensitivity

To elucidate the selective potentiation of the KCa 2.2a channels but not KCa 3.1 channels (Hougaard et al., 2007), we superimposed CyPPA into the KCa 3.1 channel cryo-EM structure. The superimposed site is defined by the R355 residue equivalent to K467 in KCa 2.2a channels and the M311 residue equivalent to V420 in KCa 2.2a channels. The V-to-M and K-to-R changes are both conservative substitutions considering their hydrophobicity and/or charge. However, the bulkier M311 and R355 residues might have forced CyPPA to sterically clash with K312 equivalent to K421 in KCa 2.2a channels. The superimposed CyPPA molecule would be only 2.0 Å from the ε-nitrogen of K312, indicating potential steric clash (Figure 4A). We then set out to render the CyPPA sensitivity to the KCa 3.1 channel through substituting residues in its proximal C-terminus for their equivalent residues in the KCa 2.2a channels. The mutations tested did not change the apparent Ca2+ sensitivity compared to the WT KCa 3.1 channels (Figure S4A, B). The responses of the WT KCa 3.1 channel to CyPPA were very minimal compared to the maximal current induced by 10 μM Ca2+ (Figure 4B). In contrast, CyPPA concentration-dependently potentiated the KCa 3.1_R355K mutant channel (Figure 4C). To compare the different magnitude of responses to CyPPA, the maximal channel currents induced by 10 μM Ca2+ were often used to normalise responses to positive modulators (I / Imax (%)) (Hougaard et al., 2007). Within the mutations tested, only KCa 3.1_R355K enabled substantial responses of the channel to CyPPA, with an EC50 value of 29.93 ± 1.03 μM (Figure 4D).

FIGURE 4. KCa 3.1 channels became sensitive towards CyPPA with a mutation in the HB helix.

FIGURE 4

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(A) CyPPA (salmon) is superimposed into the KCa 3.1 channel between the HA/HB helices (green) and CaM C-lobe (grey). (B) Limited responses to CyPPA of the WT KCa 3.1 channel. (C) Representative current traces of concentration-dependent potentiation by CyPPA of the mutant KCa 3.1_R355K channel. (D) Responses to CyPPA of the WT and mutant KCa 3.1 channels carrying mutations in the HA/HB helices. The responses were normalised by the maximal currents induced by 10 μM Ca2+.

3.5 |. Mutagenesis of the proximal C-terminus of KCa 2.1 channels to render CyPPA sensitivity

Next, we addressed why CyPPA potentiates KCa 2.2a channels but not KCa 2.1 channels (Hougaard et al., 2007). Unlike KCa 3.1 channels, KCa 2.1 channels share a high level of similarity in amino acid sequence of the CaMBD with KCa 2.2a channels (Figure 5A). A close examination of the amino acid sequences at the HB helix revealed a three-residue “AQK” insertion at residues 434 to 436 of KCa 2.1 compared with the KCa 2.2a channels (highlighted in blue, Figure 5A). This is reminiscent of a KCa 2.2 channel splice variant, KCa 2.2b (SK2b), that we reported previously (Zhang, Abrams, et al., 2012). KCa 2.2b has a “ARK” insertion at position 463 to 465, which is equivalent to the “AQK” insertion in the KCa 2.1 channel (Figure 5A). This KCa 2.2b splice variant exhibited reduced sensitivity to Ca2+ (Figure S5A, B), consistent with our previous report (Zhang, Abrams, et al., 2012) that has been independently replicated (Scholl, Pirone, Cox, Duncan, & Jacob, 2014). As a result, the responses of KCa 2.2b channels to CyPPA were recorded in the presence of 0.3 μM Ca2+, while the potentiation by CyPPA of KCa 2.2a channels were recorded in the presence of 0.15 μM Ca2+. In contrast to the KCa 2.2a channel that lacks the ARK insertion, the KCa 2.2b channel exhibited little response to CyPPA (Figure 5B).

FIGURE 5. KCa 2.1 channels became sensitive towards CyPPA with deletion mutations in the HB helix.

FIGURE 5

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(A) Amino acid sequence alignment of human KCa 2.1[GenBank: NP_002239.2] and rat KCa 2.2a [GenBank: NP_062187.1] channels at the HB helix (highlighted in green). A three-residue insertion in the middle of the HB helix is highlighted in blue. (B) Responses to CyPPA of the WT and deletion mutant KCa 2.1 channels. The responses were normalised to the maximal currents induced by 10 μM Ca2+. (C) CyPPA (salmon) is superimposed into the KCa 2.1 channel between the HA/HB helices (green) and CaM C-lobe (grey). The “AQK” three-residue insertion in the middle of HB helix is highlighted in blue.

The deletion of the three-residue “AQK” from the proximal C-terminus of KCa 2.1 channels, KCa 2.1_ΔA434/Q435/K436, did not impact its apparent Ca2+ sensitivity (Figure S5A, B). The responses of KCa 2.1 channels to CyPPA were very minimal (Figure 5B & Figure S5C), consistent with a previous report (Hougaard et al., 2007). The deletion mutant KCa 2.1_ΔA434/Q435/K436, however, exhibited concentration-dependent potentiation by CyPPA, with an EC50 value of 29.18 ± 2.78 μM (Figure 5B & Figure S5D). In the KCa 2.1 channel, the three residues A434, Q435 and K436 form an almost complete helical turn (shown in blue, Figure 5C) in the HB helix, right upstream of the cognate R438 residue. The superimposed CyPPA molecule would be only 2.3 Å from the ε-nitrogen of K392, indicating potential steric clash (Figure 5C). In the deletion mutant KCa 2.1_ΔA434/Q435/K436, the original K441 residue would move upstream and become the new K438, which may explain the sensitivity of KCa 2.1_ΔA434/Q435/K436 towards CyPPA (Figure 5B).

3.6 |. Conformational changes induced by CyPPA in KCa 2.2a channels

To elucidate any potential conformational changes induced by CyPPA in KCa 2.2a channels, we undertook molecular dynamic (MD) simulations to compare the conformational dynamics of apo KCa 2.2a channels (KCa 2.2a_apo) and the CyPPA-bound KCa 2.2a channels. There are four potential binding sites for CyPPA in the tetrameric channel structure. We docked one CyPPA molecule (KCa 2.2a_1_CyPPA), two CyPPA molecules in the opposite subunits (KCa 2.2a_2_CyPPA), three CyPPA molecule (KCa 2.2a_3_CyPPA) or four CyPPA molecules (KCa 2.2a_4_CyPPA) in all four subunits, then performed pilot MD simulations of 200 ns. During the 200 ns simulation time, all five structures have stabilised and the root-mean-square deviation (RMSD) curves have reached a plateau (Fig. S6A). We measured the minimum distance at the cytoplasmic gate of the KCa 2.2a_apo, KCa 2.2a_1_CyPPA, KCa 2.2a_2_CyPPA, KCa 2.2a_3_CyPPA and KCa 2.2a_4_CyPPA structures during the 200 ns simulation time (Fig. S6B, C, D, E, F). The minimum distance at the cytoplasmic gate is defined by a valine residue in the transmembrane S6 domain conserved among the four KCa 2 and KCa 3.1 channel subtypes (Figure S7). In the KCa 3.1 channel cryo-EM structures, this cytoplasmic gate is the distance between the V282 residues from two opposite subunits. This distance in the conductive activated state II (PDB: 6CNO) is ~3.3 Å larger than the non-conductive activated state I (PDB: 6CNN) (Lee & MacKinnon, 2018). In the KCa 2.2a channel the equivalent conserved valine residue is V391. As shown in the representative images of the cytoplasmic gate of KCa 2.2a_apo and KCa 2.2a_2_CyPPA simulations (Fig. 6A, B), we measured the minimum distances between the hydrogen atoms of the V391 residues from opposite channel subunits for both directions and then calculated the average of the two distances from each snapshot. In the KCa 2.2a_2_CyPPA simulation, the distances between the two subunits docked with CyPPA (KCa 2.2a_2_CyPPA_A) are more prominently enlarged, compared to the distances between the two subunits without CyPPA (KCa 2.2a_2_CyPPA_B) (Fig. S6D). This implies an asymmetric cytoplasmic gate in a rectangular-like shape for KCa 2.2a_2_CyPPA. As such, we used the average of the two distances (KCa 2.2a_2_CyPPA_average) from each snapshot for subsequent comparisons. The average minimum distances at the cytoplasmic gate for KCa 2.2a_1_CyPPA and KCa 2.2a_3_CyPPA are 3.89 ± 0.40 and 4.04 ± 0.31 Å (mean ± SD), both of which are wider compared to 3.42 ± 0.23 and 3.40 ± 0.25 Å (mean ± SD) of KCa 2.2a_apo and KCa 2.2a_4_CyPPA, respectively. A histogram fitted to Gaussian distribution demonstrates the widest cytoplasmic gate of the KCa 2.2a_2_CyPPA structure with an average minimum distance of 4.39 ± 0.27 Å (mean ± SD) among the five MD simulations (Figure S6B, C, D, E, F).

FIGURE 6. CyPPA enlarged the minimum distance at the cytoplasmic gate of the KCa 2.2a channel in MD simulations.

FIGURE 6

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(A) Representative images of the cytoplasmic gate of KCa 2.2a_apo simulations. (B) Representative images of the cytoplasmic gate of KCa 2.2a_2_CyPPA simulations. The minimum distances between the hydrogen atoms of the V391 residues from opposite channel subunits for both directions were measured and then the average of the two distances was calculated from each snapshot. The minimum distance at the cytoplasmic gate is shown as a function of the simulation time for (C) two CyPPA molecules in the opposite subunits (KCa 2.2a_2_CyPPA), (D) apo (KCa 2.2a_apo) and (E) four CyPPA molecules (KCa 2.2a_4_CyPPA) in all four subunits of KCa 2.2a channels. The distribution histogram of the average minimum distance at the cytoplasmic gate shows a much larger distance in the KCa 2.2a_2_CyPPA simulations (F) compared with the KCa 2.2a_apo (G) and KCa 2.2a_4_CyPPA (H) simulations.

We performed another round of MD simulations of the KCa 2.2a_apo, KCa 2.2a_2_CyPPA and KCa 2.2a_4_CyPPA structures with simulation time of 200 ns. The summary of the two rounds of 200 ns simulations are shown in Figure S8. A wider cytoplasmic gate was consistently observed in the simulations of the KCa 2.2a_2_CyPPA structure, compared to the simulations of the KCa 2.2a_apo and KCa 2.2a_4_CyPPA structures (Figure S9).

In the final simulations of 500 ns, all structures have stabilised and the RMSD curves have reached a plateau (Fig. S10). The average minimum distances at the cytoplasmic gate of KCa 2.2a_2_CyPPA (Figure 6C) were consistently larger than KCa 2.2a_apo (Figure 6D) and KCa 2.2a_4_CyPPA (Figure 6E) during the 500 ns simulation time. A histogram fitted to Gaussian distribution demonstrates a much wider cytoplasmic gate of the KCa 2.2a_2_CyPPA structure with an average minimum distance of 4.80 ± 0.34 Å (mean ± SD, Figure 6F), compared to 3.51 ± 0.44 and 3.54 ± 0.23 Å (mean ± SD) of the KCa 2.2a_apo (Figure 6G) and KCa 2.2a_4_CyPPA (Figure 6H), respectively. KCa 2.2a_2_CyPPA induced a ~1.29 Å increase in the cytoplasmic gate of the KCa 2.2a channel, echoing the positive modulation of KCa 2.2a channels by CyPPA.

4 |. Discussion and Conclusions

CyPPA emerged as a prototype subtype-selective modulator that increases the apparent Ca2+ sensitivity of KCa 2.2a and KCa 2.3 channel but not that of the KCa 2.1 and KCa 3.1 channels (Hougaard et al., 2007). Our previous crystallography study showed a putative binding site for the non-selective KCa 2 channel positive modulator, 1-EBIO between the N-lobe of CaM and the CaMBD (Zhang, Pascal, et al., 2012). Independently of our work, a Pfizer group revealed a putative binding site of the KCa 2.2a / KCa 2.3-selective CyPPA analog between the N-lobe of CaM and the CaMBD (Cho et al., 2018). In the full-length KCa 3.1 channels, the N-lobe of CaM actually interacts with the S4-S5 linker, instead of the CaMBD (Lee & MacKinnon, 2018). The putative binding site of 1-EBIO (Zhang, Pascal, et al., 2012) and the CyPPA analog (Cho et al., 2018) between the N-lobe of CaM and the CaMBD may not exist in the full-length KCa 2 channel, because the N-lobe of CaM interacts with the S4-S5 linker in the full-length structure. The S4-S5 linker was demonstrated as a critical element in the KCa 3.1 channel to confer Ca2+ sensitivity (Lee & MacKinnon, 2018). This interface between the CaM N-lobe and the S4-S5 linker was then explored by mutagenesis as the potential binding pocket of an KCa 3.1-selective modulator (e.g. SKA-111) (Shim et al., 2019).

If KCa 3.1 channel selective modulators bind to the interface between the CaM N-lobe and the S4-S5 linker, then what about CyPPA, the selective modulator for KCa 2.2a and KCa 2.3 channels? The S4-S5 linker may not contribute to the subtype-selectivity of this modulator, as swapping chimeras and mutations of the S4-S5 linker did not influence the responses of KCa 2.2a channels to CyPPA (Figure 1). Mutating residues in the HA (V420M) and HB (K467R) helices of KCa 2.2a channels to their equivalent residues in KCa 3.1 channels led to decreased potency of CyPPA (Figure 3C, D), while substituting a residue in the HB helix (R355K) of KCa 3.1 channels rendered its sensitivity to CyPPA (Figure 4C, D), indicating a crucial role of the HA/HB helices in the subtype-selectivity of CyPPA. The reason why CyPPA selectively potentiates KCa 2.2a channels but not KCa 2.1 channels may lie in the fact that KCa 2.1 channels harbors a three-residue insertion (A434/Q435/K436) in its HB helix, right upstream of the cognate arginine residue (R438) equivalent to K467 of KCa 2.2a channels. The deletion mutant KCa 2.1_ΔA434/Q435/K436 became sensitive to CyPPA (Figure 5B). Collectively, these mutagenesis results on KCa 2.1, KCa 2.2a and KCa 3.1 channels established the HA/HB helices as a structural determinant for CyPPA’s subtype-selectivity. Mutations of several residues including Q470, N474, A477, L480 and V481 changed the response of KCa 2.2a channels to CyPPA or its analog in previous reports (Cho et al., 2018; Zhang, Pascal, et al., 2012). None of these residues is within 5 angstroms from the CyPPA docked between CaM C-lobe and the HA/HB helices. Therefore, they are unlikely to interact with CyPPA directly. All these residues are in the HB helix, facing the almost parallel HA helix (Figure S11). Mutating these residues could potentially cause conformational changes at the interface between HA and HB helices, which may indirectly influence the CyPPA binding at the other end of the HA/HB helices.

With CyPPA docked between CaM C-lobe and the HA/HB helices, we performed MD simulations. There are four potential binding sites for CyPPA in the tetrameric channel structure. It is unknown how many binding sites need to be occupied by CyPPA to potentiate the channel activity. In MD simulations, when one or three potential sites are occupied by CyPPA, the cytoplasmic gate was mildly widened compared to the apo channel simulations (Figure S6). When CyPPA occupied the two binding sites in two opposite channel subunits, the cytoplasmic gate was enlarged the most (Figure S6 and Figure 6). These changes are smaller than the difference of ~3.3 Å between the conductive activated state II and non-conductive activated state I of KCa 3.1 channels (Lee & MacKinnon, 2018). Thus, the simulations may represent a state that is easier to open rather than the fully conductive state, echoing CyPPA as a positive allosteric modulator of KCa 2.2a channels (Hougaard et al., 2007). This smaller change in the cytoplasmic gate may also reflect the smaller single channel conductance of KCa 2.2a compared with KCa 3.1 channels (Aldrich et al., 2019).

Unlike NS309 that potentiates KCa 2.2a channel activity to ~100% of the maximal current on the human KCa 2.2a channels, the Emax of CyPPA is ~71% (Hougaard et al., 2007). In our hands, the Emax induced by 200 μM CyPPA is 81.17 ± 4.30 % on the rat KCa 2.2a channels (Figure 1). We did not observe inhibition of KCa 2.2a channels at this saturating concentration of CyPPA in the bath solution. But we do not know whether CyPPA would potentiate or inhibit KCa 2.2a channel activity at even higher concentrations than 200 μM, if CyPPA were more water-soluble. In MD simulations, occupation of all four potential sites in KCa 2.2a channels by CyPPA did not change the cytoplasmic gate compared to the apo channel simulations (Figure S6 and Figure 6). One speculation is that channels bound with one, two or three CyPPA molecules are potentiated, while channels with four binding sites occupied by CyPPA are not, which leads to the ~70–80% efficacy. Another possibility is that more water-soluble analogs of CyPPA would potentiate KCa 2.2a channel activity at low concentrations but inhibit channel activity at very high concentrations, which will require the development of such analogs for future studies.

A close examination of the KCa 2.2a_2_CyPPA simulations revealed an asymmetrical cytoplasmic gate. This is reminiscent of the crystal structure of a constitutively active R201A mutant of Kir3.2 channel (Whorton & MacKinnon, 2011). Two PIP2 molecules bind to two opposite subunits of the tetrameric Kir3.2_R201A channel and enlarge the G-loop gate only between those two opposite subunits (PDB code: 3SYQ). A gate with a rectangular-like shape may require lower energy than a gate in a fully open model and thus more often observed in MD simulation studies (Meng, Zhang, Logothetis, & Cui, 2012). The functional significance of this asymmetrical gate in KCa channels will require future structural studies similar to what has been done with the Kir channels. Nonetheless, the fact that the distances at cytoplasmic gate between the two subunits docked with CyPPA (KCa 2.2a_2_CyPPA_A) were enlarged more than the distances between the two subunits without CyPPA (KCa 2.2a_2_CyPPA_B) suggests that the changes in cytoplasmic gate are indeed induced by CyPPA binding.

KCa 2 channels are activated exclusively by intracellular Ca2+, changes in their apparent sensitivity to Ca2+ may result in abnormal neuronal excitability. An I289N mutation diminished KCa 2.2 channel activity and led to the tremor phenotypes in the tremor dominant Kyoto (Trdk) rats (Kuramoto et al., 2017), reflecting the crucial role of KCa 2 channels in the central nervous system. Since KCa 2.2 channel activity is linked with movement disorders, drugs targeting this channel subtype may become therapeutic agents for ataxia (Alvina & Khodakhah, 2010; Gao et al., 2012; Kasumu, Hougaard, et al., 2012; Walter et al., 2006), spinal muscular atrophy (Dimitriadi et al., 2013), and Parkinson’s disease (Liu, Wang, & Chen, 2010). Here, we focus on the molecular determinants for the subtype-selectivity of CyPPA. Future studies may take advantage of these results for drug development for movement disorders.

Supplementary Material

1

BULLET-POINT SUMMARY.

What is already known

  • CyPPA positively modulates KCa 2.2a and KCa 2.3, but not KCa 2.1 and KCa 3.1 channel subtypes.

What does this study add

  • CyPPA may modulate KCa 2.2a channels through interacting with the HA/HB helices.

  • V420 and K467 residues on the HA/HB helices are critical for the subtype-selectivity of CyPPA.

What is the clinical significance

  • Subtype-selective modulators of KCa 2 channels are potential therapeutic agents for movement disorders.

ACKNOWLEDGEMENTS

We thank Drs. Heike Wulff and Keykavous Parang for helpful comments on the manuscript. We are grateful to Lisa Tran, Kimberly Diep, Michelle Le and Maria Akhnoukh for technical assistance. The computations were supported by the ITS (Information Technology Services) Research Computing at Northeastern University. M.Z. was supported by a Scientist Development Grant 13SDG16150007 from American Heart Association, a YI-SCA grant from National Ataxia Foundation and a grant 4R33NS101182-03 from National Institutes of Health, USA.

FUNDING INFORMATION

This work was funded by a Scientist Development Grant 13SDG16150007 from American Heart Association, a YI-SCA grant from National Ataxia Foundation and a grant 4R33NS101182-03 from National Institutes of Health, USA.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design and Analysis and as recommended by funding agencies, publishers and other organizations engaged with supporting research.

Abbreviations:

CaM

calmodulin

CaMBD

calmodulin-binding domain

mAHP

medium afterhyperpolarization

MD

Molecular dynamics

S4-S5 linker

linker between S4 and S5 transmembrane domains

RMSD

root-mean-square deviation

SCAs

spinocerebellar ataxias

KCa 2 channels

small-conductance Ca2+-activated potassium channels

Trdk rat

tremor dominant Kyoto rat

LIST OF HYPERLINKS for GUIDE TO PHARMACOLOGY :

KCa 2.1:

https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=381&familyId=69&familyType=IC

KCa 2.2

https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=382&familyId=69&familyType=IC

KCa 2.3

https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=383&familyId=69&familyType=IC

KCa 3.1

https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=384&familyId=69&familyType=IC

CyPPA

https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2323

Footnotes

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ETHICS APPROVAL

Not applicable. No animal or human subject was involved.

DATA AVAILABILITY

The PDB file of the docked CyPPA in the KCa 2.2a channel is openly available in digital commons of Chapman University at https://digitalcommons.chapman.edu/pharmacy_data/5/. Other data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1738338-supplement-1.pdf (4.7MB, pdf)

Data Availability Statement

The PDB file of the docked CyPPA in the KCa 2.2a channel is openly available in digital commons of Chapman University at https://digitalcommons.chapman.edu/pharmacy_data/5/. Other data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

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