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.

本期《生理学报》介绍了Zhang博士及其团队的最新工作，涉及小电导Ca ²⁺激活的K ⁺（SK）通道，更确切地说是SK2亚型的激活机制。¹的确，这些仍不清楚的通道在几种类型的细胞的生理和病理中起着重要作用。

SK通道由四个不同的基因编码，即SK1的KCNN1（KCa2.1），SK2的KCNN2（KCa2.2），SK3的KCNN3（KCa2.3）和SK4的KCNN4（IK或KCa3.1）。它们在整个四聚体装配中类似于电压门控钾通道，因为它们由每个SK通道亚基中的六个跨膜结构域（编号为S1-S6）组成。但是，它们在这些小电导通道对电压不敏感的意义上有所不同，它们由细胞内低浓度的钙离子激活，该钙离子与钙调蛋白结合，钙调蛋白是一种能够与钙结合的蛋白，钙蛋白自然地与细胞内SK通道结合地区。有趣的是，大电导钾（BK）通道与SK家族有关，可以被电压或细胞内钙激活。

在心血管系统中，SK通道参与内皮依赖性超极化，而在中枢神经系统中，SK通道通过中等超极化后（mAHP）降低几种神经元的兴奋性。因此，几个团队已将SK通道作为多种病理学中的药物靶标。^2-7为了利用这种新的治疗选择，需要靶向SK通道的小分子。

在由Nam等人发表本文件中，“HA螺旋和S4-S5接头调制表观钙之间的疏水相互作用²⁺ SK2通道的敏感性”，¹他们报告交换机的分子决定簇的到导电状态了广泛的研究开始由Ca 2 ^+-钙调蛋白复合物。

以前，描述了改变表观Ca ²⁺敏感性的正调节剂，即1-EBIO和NS309，并研究了它们在钙调蛋白结合位点的作用机理。^[8，9 ]这种配体似乎并没有改变钙离子对钙调蛋白的实际亲和力，而是改变了与通道复合物门控相关的信号传递。

由于cryo-EM技术的最新发展，出现了研究门控机制的新可能性，并由此开发了新型调制器。实际上，IK通道，相关的SK通道（也称为SK4）以及BK通道已使用此技术进行了结构解析。^10-12

正如作者在本研究中所做的那样，这一进展允许为SK2通道构建更好的同源性模型。

该模型仍然缺少生理条件下存在的天然相关蛋白。但是，它可以有利地用于破译与孔的打开和关闭有关的机构。此外，这种结构信息可能是理解化合物在该区域相互作用的作用方式的关键。

在先前的工作中，作者发现钙调蛋白结合结构域（V407F）附近的细胞内V至F突变导致对表观钙浓度的敏感性增加了六倍的结构。¹³他们将这种增加归因于氨基酸取代引起的通道结构的强烈变化，从而引起疏水相互作用。这种V至F突变也已经用于研究相同通道的外孔区域中其他配体的结合机理。实际上，由于较高的疏水性相互作用，SK2和SK3中的V366F突变和V520F突变分别产生了与TEA（一种众所周知的离子通道孔阻断剂）的亲和力，从而大大提高了亲和力[ ^14，15]。同时还显着降低了对木瓜蛋白酶的亲和力，后者是SK家族¹⁴的参照阻滞剂，被发现与通道的外孔区域结合。^16-18

除了这项体外研究外，作者还报告了对秀丽隐杆线虫的一项体内研究，该研究表明，kcnl-2通道（SK2的直系同源通道）中的等效苯丙氨酸突变可减少现有ALS模型中的运动缺陷，显示出潜在的影响这种突变的细胞功能在体内得以维持。¹关于体内突变的后果，SK3通道中罕见的S436C突变导致称为Zimmermann-Laband综合征的疾病，影响牙龈，指甲和面部特征等多种组织的发育。¹⁹与Nam的论文中观察到的相似，这种位于钙调蛋白与通道的一个亚基之间的界面处的突变诱导了相应通道的更快，更完全的激活。实际上，用半胱氨酸¹⁹替代丝氨酸436引起局部疏水性的增加和形成氢键的倾向降低，从而导致结构变化也影响门控。

在这里，Nam等人探索了SK2通道钙敏感性的新决定因素区域，该区域决定了其开放。他们使用了由于最近的cryo-EM发现而获得的更新模型，他们在目标位点的空间附近发现了一部分氨基酸残基，这些残基可能参与了钙与通道复合物的结合所引起的涌入的传导。实际上，E404-M412段将形成一个名为HA螺旋的螺旋，该螺旋与另外两个名为S ₄₅ A和S ₄₅ B的螺旋紧密接触。由于该HA螺旋中的氨基酸残基具有疏水性，因此被证明可以修饰通过先前的V到F突变，SK通道中的钙敏感性，他们探索了新发现的相关S ₄₅ A和S ₄₅B段。

丙氨酸替代非极性氨基酸I289，I295，F297和F301有效降低了Ca ²⁺的表观敏感性。相反地​​，在该区域附近突变N293极性残基以获得更多的疏水性亮氨酸或苯丙氨酸会增加灵敏度。相反，分别通过赖氨酸和天冬氨酸突变获得的该位置的正电荷和负电荷都破坏了钙的活化。

为了完成他们的研究，他们检查了SK4通道与SK2的差异，其中与以前研究过的SK2中的V407F对应的V298F突变并没有显着影响钙敏感性。

因此，他们创建了两个嵌合通道。首先，一个SK2通道包含SK4的S4-S5段，而互补的SK4通道包含SK2的S4-S5段。这两个通道都起作用。新的嵌合SK4通道中的V298F突变确实导致了表观钙敏感性的增加，就像SK2等效VF突变体中一样，嵌合SK2通道中的V407F突变具有降低表观钙敏感性的反作用。这些实验精美地证明了，SK通道的HA螺旋与相邻的S4-S5段之间的相互作用是钙依赖性激活通道的关键决定因素。

最后，研究小组创建了一个SK2双突变体，结合了两个突变，这些突变导致钙敏感性增加：HA螺旋中的V407F和S4-S5段中的N293A。如所期望的，该SK2突变体表现出对Ca ²⁺的最高测量的表观敏感性。

为了探索这些突变在细胞质门水平的影响，作者对WT SK2和V407F / N293A双突变体的同源性模型进行了分子动力学模拟。这导致测量V282与形成该通道门的相对亚基之间的平均距离略有增加（0.18埃），为该研究中观察到的Ca ²⁺敏感性变化提供了潜在的分子机制。

总之，以前的SK通道阳性调节剂（如1-EBIO和NS309）具有干扰SK通道钙敏感性的治疗潜力。这项工作进一步证明了这一点，并为进一步发展奠定了基础，这可能会导致治疗多种疾病和疾病的新颖医学选择。为了改进这种作用方式，应使这种调节亚型特异性。根据涉及的SK亚型SK2或SK3，这将允许靶向特定的病理学，并且由于涉及较少的细胞靶标，因此将减少这种治疗的可能的副作用。