Deciphering the molecular mechanism of SK2 channel activation by intracellular calcium to develop new therapeutic agents

This issue of Acta Physiologica presents the latest work of Dr Zhang and his team concerning the mechanism of activation of small-conductance Ca2+-activated K+ (SK) channels, and more precisely the SK2 subtype.1 Indeed, these still poorly understood channels play important roles in the physiology and the pathology of several types of cells. SK channels are encoded by four different genes, namely KCNN1 for SK1 (KCa2.1), KCNN2 for SK2 (KCa2.2), KCNN3 for SK3 (KCa2.3) and KCNN4 for SK4 (IK or KCa3.1) channels. They resemble voltage-gated potassium channels in their overall tetrameric assembly as they are composed of six transmembrane domains, numbered S1-S6, in each SK channel subunit. However, they differ in the sense that these small conductance channels are insensitive to voltage, they are instead activated by low intracellular concentrations of calcium ions which bind to calmodulin, a protein capable of attaching to calcium that is naturally bound to SK channels in the intracellular region. It is interesting to note that the big conductance potassium (BK) channel is related to the SK family and can be activated by either voltage or intracellular calcium. In the cardiovascular system, SK channels participate in endothelium-dependent hyperpolarization, while in the central nervous system, they reduce the excitability of several types of neurons through the medium AfterHyperPolarization (mAHP). Hence, SK channels have been proposed as drug targets in multiple pathologies by several teams.2-7 There is a need for small-molecules targeting SK channels in order to exploit this new therapeutic option. In the present paper published by Nam et al, “Hydrophobic interactions between HA helix and S4-S5 linker modulate apparent Ca2+ sensitivity of SK2 channels”,1 they report an extensive investigation of the molecular determinants for the switch to a conductive state initiated by the Ca2+-calmodulin complex. Previously, positive modulators modifying the apparent Ca2+ sensitivity were described, namely 1-EBIO and NS309, and their mechanism of action at the calmodulin binding site had been investigated.8, 9 It seems that this type of ligand does not alter the actual affinity of calcium ions for calmodulin, but they rather modify the transmission of the signal caused by such a link to the gating of the channel complex. Thanks to the recent development of the cryo-EM technique, new possibilities for the investigation of gating mechanisms arose, and with that, for the development of novel modulators. Indeed, the IK channel, a related SK channel also known as SK4, as well as the BK channel have been structurally resolved using this technique.10-12 This advancement allowed for the construction of a better homology model for SK2 channels as the authors did in the present study. The model still lacks naturally associated proteins that are present in physiological conditions; nevertheless, it can be used advantageously to decipher the mechanisms involved in the opening and closing of the pore. Furthermore, such structural information can be the key for understanding the mode of action of compounds interacting in this region. In a previous work, the authors found that a V to F mutation, intracellularly, in proximity of the calmodulin binding domain (V407F) leads to a structure with a sixfold increased sensitivity to apparent calcium concentration.13 They attributed this augmentation to hydrophobic interactions through strong changes in the channel structure caused by the amino acid substitution. Such V to F mutation has also been used to investigate the binding mechanism of other ligands in the outer pore region of the same channels. Indeed, the V366F mutation and the V520F mutation, in SK2 and SK3, respectively, yielded strongly increased affinity with TEA, a well-known pore blocker of ion channels, because of higher hydrophobic interactions,14, 15 while also significantly decreasing the affinity for apamin, the blocker of reference for the SK family14 which was found to bind in the outer pore region of the channels.16-18 In addition to this in vitro study, the authors report an in vivo investigation in C elegans which shows that an equivalent phenylalanine mutation in kcnl-2 channels, an orthologous channel to SK2, reduce locomotion defects in an existing ALS model, showing the potential impact on cell function of this mutation is maintained in vivo.1 Regarding the consequences of in vivo mutations, a rare S436C mutation in SK3 channels leads to a disease called the Zimmermann-Laband syndrome affecting the development of many types of tissue such as gums, nails and facial features.19 This mutation located at the interface between calmodulin and one of the channel's subunits induces a faster and more complete activation of the corresponding channel similarly to what is observed in the Nam's paper. Indeed, the replacement of the serine 436 by a cysteine19 induces an increase of the local hydrophobicity and reduced propensity to create hydrogen bond leading to structural changes also affecting the gating. Here, Nam et al explore new determinant regions of SK2 channel's calcium sensitivity responsible for its opening. Using updated models obtained thanks to the recent cryo-EM findings, they identified a stretch of amino acid residues in spatial proximity of the site of interest that could participate in the conduction of the influx caused by the binding of calcium to the channel complex. Indeed, the E404-M412 segment would form a helix named HA helix, which is in close contact with two other helices, named S45A and S45B. As the hydrophobicity of amino acid residues in this HA helix was proven to modify apparent calcium sensitivity in SK channels through the previous V to F mutations, they explored the newly found associated S45A and S45B segments. The replacement of apolar amino acids I289, I295, F297 and F301 by alanine effectively reduced the apparent Ca2+ sensitivity. Adversely, mutating the N293 polar residue in close vicinity of this region for more hydrophobic leucine or phenylalanine increased the sensitivity. In contrast, both positive and negative charges in that location, obtained with lysine and aspartate mutations, respectively, disrupted the calcium activation. To complete their investigation, they inspected the difference of SK4 channels, when compared to SK2, in which the V298F mutation, corresponding to the previously studied V407F in SK2, did not significantly influence calcium sensitivity. Therefore, they created two chimeric channels. First, a SK2 channel containing the S4-S5 segment of SK4, and the complementary SK4 channel containing the S4-S5 segment of SK2. Both these channels were functional. The V298F mutation in the new chimeric SK4 channel did lead to increased apparent calcium sensitivity as in the SK2 equivalent VF mutant, and the V407F mutation in the chimeric SK2 channel had the inverse effect of reducing apparent calcium sensitivity. These experiments beautifully proved that the interaction between the HA helix of SK channels with the neighbouring S4-S5 segment is a crucial determinant of calcium dependent activation of the channel. Finally, the team created a SK2 double mutant, combining both mutations that yielded increases in calcium sensitivity: V407F in the HA helix and N293A in the S4-S5 segment. As expected, this SK2 mutant exhibited the highest measured apparent sensitivity towards Ca2+. To explore the effect of these mutations at the cytoplasmic gate level, the authors performed molecular dynamic simulations on homology models of the WT SK2 and the V407F/N293A double mutant. This lead to the measure of a slight increase (0.18 angström) of the mean distance between the V282 from opposite subunits that form the gate of this channel, providing a potential molecular mechanism for the variation of the Ca2+ sensitivity observed in this study. In conclusion, previous positive modulators of SK channels like 1-EBIO and NS309 established the therapeutic potential of interfering with the calcium sensitivity of SK channels. This work further proves this point and sets the basis for further development that could lead to novel medical options to treat several conditions and diseases. In order to refine this mode of action, such modulation should be made subtype specific. This would allow to target specific pathologies, depending on the SK subtype that is involved, SK2 or SK3, and would reduce the possible side effects of such treatment as fewer cellular targets would be involved. The authors declare no conflict of interest.