Subtype-Selective Positive Modulation of KCa2.3 Channels Increases Cilia Length

Abstract

Small-conductance Ca²⁺-activated potassium (K_Ca2.x) channels are gated exclusively by intracellular Ca²⁺. The activation of K_Ca2.3 channels induces hyperpolarization, which augments Ca²⁺ signaling in endothelial cells. Cilia are specialized Ca²⁺ signaling compartments. Here, we identified compound 4 that potentiates human K_Ca2.3 channels selectively. The subtype selectivity of compound 4 for human K_Ca2.3 over rat K_Ca2.2a channels relies on an isoleucine residue in the HA/HB helices. Positive modulation of K_Ca2.3 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 K_Ca2.3_G351D mutant channels and the reduced cilia length resulting from the expression of Ca²⁺-hyposensitive K_Ca2.3_I438N channels. Collectively, we were able to associate functions of K_Ca2.3 channels and cilia, two crucial components in the flow-induced Ca²⁺ signaling of endothelial cells, with potential implications in vasodilation and ciliopathic hypertension.

Figure 1

Positive modulation of human K_Ca2.3 channels by compounds. (A) Chemical structures of compounds 2k–2v, 3a–3g, and 4, compared with those of CyPPA and NS13001. (B) Concentration-dependent potentiation of K_Ca2.3 channels by compounds. (C) EC₅₀ values to compounds of K_Ca2.3 channels. (D) Responses to compounds of K_Ca2.3 channels were normalized to the maximal currents induced by 10 μM Ca²⁺. (E) E_max to compounds of K_Ca2.3 channels. The numbers of independent recordings are shown in parentheses for CyPPA (8), NS13001 (5), 2m (5), 2n (5), 2p (5), 2r (5), 2s (6), 2t (7), 2v (6), and 4 (7). Data are presented as mean ± SD.

Figure 2

Subtype selectivity of compound 4 relies on the HA/HB helices. (A) Amino acid sequence alignment of rat K_Ca2.2a [GenBank: NP_062187.1], human K_Ca2.3 [GenBank: NP_002240.3], and human K_Ca3.1 [GenBank: NP_002241.1] channels at the proximal C terminus. HA and HB helices are highlighted in green. I568 in K_Ca2.3 channels and their equivalent residues are shown in bold. (B) Potentiation by compound 4 of the WT and mutant human K_Ca2.3 channels. (C) EC₅₀ values for potentiation by compound 4. ***P < 0.001 compared with human K_Ca2.3_WT. (D) Responses to compound 4 were normalized to the maximal currents induced by 10 μM Ca²⁺. (E) E_max to compound 4 of the WT and mutant K_Ca2.3 channels. The numbers of independent recordings are shown in parentheses for K_Ca2.3_WT (7) and K_Ca2.3_I568V (6). Data are presented as mean ± SD.

Figure 3

The effects of K_Ca2.3 channel potentiation by compound 4 on cilia length in ET cells. (A) Cells were stained with the antibody of the ciliary marker acetylated α-tubulin (green) and the nuclear marker (DAPI; blue). (B) Cilia length was grouped in a discreet range, and percent distribution was tabulated. (C) Cilia length is significantly longer in cells treated with the positive modulator, compound 4 (20 μM). N = 50–70 for each slide preparation, and a total of four independent slides were used in each group. Data are presented as mean ± SD. *p < 0.05 compared to the control.

Figure 4

Mutant mouse K_Ca2.3 channels with altered Ca²⁺ sensitivity. Mutations channels were expressed in ET cells and their apparent Ca²⁺ sensitivity was evaluated using inside-out patch clamp recordings. (A) Representative K_Ca2.3_WT channel currents in response to Ca²⁺. (B) Concentration-dependent activation by Ca²⁺ of the mutant and WT K_Ca2.3 channels. (C) EC₅₀ values to Ca²⁺ of the mutant and WT K_Ca2.3 channels. The numbers of independent recordings are shown in parentheses for K_Ca2.3_WT (6), K_Ca2.3_G351D (7), and K_Ca2.3_I438N (5). Data are presented as mean ± SD. ***P < 0.001 compared with K_Ca2.3_WT.

Figure 5

Expression of mouse K_Ca2.3 channels changes the primary cilia length in ET cells. (A) Cells were stained with the antibody of the ciliary marker acetylated α-tubulin (green) and the nuclear marker DAPI (blue). (B) Cilia length was grouped in a discreet range, and percent distribution was tabulated. (C) Cilia length is significantly longer in cells expressing K_Ca2.3_WT and K_Ca2.3_G351D but shorter in cells expressing K_Ca2.3_I438N channels. N = 50–70 for each slide preparation, and a total of four independent slides were used in each group. Data are presented as mean ± SD. *p < 0.05 and ****p < 0.0001 compared to the control.

Figure 6

Positive and negative modulation of K_Ca2.3 channels affected flow-induced cytosolic Ca²⁺ signaling. Fluorescence Ca²⁺ measurements of ET cells treated with (A) solvent control, (B) negative modulator AP14145 (20 μM), and (C) positive modulator compound 4 (20 μM). (D) Peak Ca²⁺ values are significantly increased by compound 4 but reduced by AP14145. The numbers of independent measurements are shown in parentheses for the control (5), AP14145 (5), and compound 4 (5). Data are presented as mean ± SD. *p < 0.05 and ****p < 0.0001 compared with the control.