A single coiled‐coil domain mutation in hIKCa channel subunits disrupts preferential formation of heteromeric hSK1:hIKCa channels

Figures

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  FIGURE 1. Generation of a pore mutant hSK1 subunit produced a current sensitive to both apamin and TRAM‐34. (ai) Current–voltage (IV) relationship for hSK1[S348T + A371V] generated by a voltage ramp from −100 to +100 mV (1 s duration) in the absence and presence of apamin (100 nM). The Ca2+‐dependent inward rectifying current was completely inhibited by apamin leaving a linear IV relationship. (aii) IV relationship for hSK1[S348T + A371V] in the absence and presence of TRAM‐34 (10 μM), showing the mutant hSK1 channel current was sensitive to the hIKCa inhibitor. (b) Diary plot showing amplitude of channel currents over time. Amplitude of hSK1‐ and hSK1:hIKCa‐mediated currents were stable, while current rundown was observed for both homomeric and heteromeric channels containing the hSK1[S348T + A371V] subunit. Homomeric hSK1[S348T + A371V]‐mediated current decreased in amplitude with a τ of 2 min (fit shown as solid line). Inhibition of hSK1[S348T + A371V]‐mediated current by TRAM‐34 (10 μM) was resolved by early application, causing inhibition of current with a τ of 20 s (fit shown as solid line).

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  FIGURE 2. Expression of hSK1[S348T + A371V] with WT IKCa subunits produced a current that was insensitive to apamin but inhibited by TRAM‐34. (a) IV relationship generated by a voltage ramp from −100 to +100 mV (1 s duration) in the absence and presence of apamin (100 nM), showing that the heteromeric channel current is insensitive to the SK channel inhibitor. (b) IV relationship in the absence and presence of TRAM‐34 (10 μM), showing the current recorded from cells expressing both hSK1[S348T + A371V] and WT IKCa subunits was fully sensitive to the hIKCa inhibitor.

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  FIGURE 3. Comparison of sensitivities to inhibition by TRAM‐34. (a) Concentration–inhibition relationships generated by inhibition of expressed channel currents by increasing concentrations of TRAM‐34. Homomeric hIKCa‐mediated current was most sensitive and fully blocked by TRAM‐34. Current from cells expressing hSK1[S348T + A371V] and WT IKCa subunits was also fully sensitive to TRAM‐34, with an intermediate sensitivity between homomeric hIKCa‐ and heteromeric hSK1:hIKCa‐mediated current. (b) Mean IC50 values obtained from inhibition of each current subtype, with significance determined by one way ANOVA.

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  FIGURE 4. Expression of the hIKCa(H389E) subunit produced functional channels. (ai) IV relationship of expressed hIKCa[H389E]‐mediated current evoked by a voltage ramp from −100 to +100 mV (1 s duration) in the absence and presence of TRAM‐34 (10 μM). Evoked current was fully sensitive to TRAM‐34 (n = 3) and is shown in comparison with the effect of TRAM‐34 (10 μM) on WT IKCa‐mediated current (n = 6) (aii). (bi) Comparison of inhibition evoked by addition of 10 μM TRAM‐34 of both WT hIKCa‐ and hIKCa[H389E]‐mediated current. (bii) Comparison of excised outside‐out patch current amplitude measured at −60 mV for hIKCa(H389E) (n = 3) and WT hIKCa (n = 8) channels indicating comparable expression of both wildtype and mutant channels. Significance determined using unpaired Student's t test.

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  FIGURE 5. Sensitivities to inhibition by TRAM‐34 of homomeric and heteromeric channel currents. (a) IV relationships for hIKCa (left), hSK1:hIKCa (centre) and hSK1:hIKCa(H389E) (right) evoked currents in the absence and presence of increasing concentrations of TRAM‐34. (b) Concentration–inhibition relationships for the effect of TRAM‐34 on homomeric hIKCa, heteromeric hSK1:hIKCa and heteromeric hSK1:hIKCa[H389E] channel currents. Homomeric hIKCa‐mediated current was fully inhibited by TRAM‐34 with an IC50 of 51.1 ± 12 nM (n = 4), while both populations of heteromeric currents were only partially inhibited by TRAM‐34. The observed IC50 values for inhibition of heteromeric channel currents were 596.8 ± 82.4 nM for hSK1:IKCa (WT) (n = 4) and 250 nM ± 53 nM (n = 3) for hSK1:IKCa[H389E]. The higher sensitivity and less steep slope observed for inhibition of hSK1:hIKCa[H389E] channel current was suggestive of a mix of both homomeric and heteromeric channels. (c) IC50 values for homomeric hIKCa‐ and heteromeric hSK1:hIKCa‐mediated current and the comparison with the IC50 value from the inhibition relationship produced from cells co‐expressing hSK1 and hIKCa[H389E] subunits.

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  FIGURE 6. Disruption of heteromeric channel assembly reveals apamin‐sensitive hSK1‐mediated current. (a) Addition of apamin (100 nM) had no effect on current resolved from cells co‐expressing wildtype hSK1 and hIKCa subunits (n = 4). (b) Addition of apamin (100 nM) partially inhibited current resolved from cells co‐expressing wildtype hSK1 and the mutant hIKCa[H389E] subunits (n = 6).

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  FIGURE 7. Homomeric hSK1‐mediated current identified by inhibition by UCL1684 resulting from disruption of heteromeric hSK1:hIKCa(H389E) channel formation. (a) IV relationships for hSK1 (left), hSK1:hIKCa (WT) (centre) and hSK1:hIKCa[H389E] (right) evoked currents in the absence and presence of increasing concentrations of UCL1684. (b) Concentration–inhibition relationships for the effect of UCL1684 on homomeric hSK1, heteromeric hSK1:hIKCa (WT) and heteromeric hSK1:hIKCa[H389E] channel currents. Homomeric hSK1‐mediated current was fully inhibited by UCL1684 with an IC50 of 783 pM (n = 3), while both populations of heteromeric currents were only partially inhibited by UCL1684. Heteromeric channels comprised of wildtype hSK1 and hIKCa (WT) subunits displayed a single component inhibition relationship with an IC50 of 236 nM (n = 3). In contrast, heteromeric channel current from cells co‐expressing wildtype hSK1 and mutant hIKCa[H389E] subunits displayed a two‐component inhibition relationship. The first component reflected inhibition of homomeric hSK1 channels (IC50 511 pM), while the second component reflected inhibition of heteromeric hSK1:hIKCa[H389E] channel current (IC50 301 nM) (n =…

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  FIGURE 8. Differing sensitivities to activation by Ca2+ of homo‐ and heteromeric channels. (a) IV relationships for hSK1‐, hIKCa‐ and hSK1:hIKCa‐evoked currents in the presence of increasing concentrations of Ca2+. (b) Ca2+ activation curves showing that homomeric hIKCa channels appeared most sensitive to activation by Ca2+ (EC50 = 409 ± 15.3 nM) (n = 5), with a relationship that demonstrated that these channels were active at resting Ca2+ levels. In contrast, both homomeric hSK1 (EC50 = 531 ± 78 nM) (n = 4) and heteromeric hSK1:hIKCa (EC50 = 613 ± 27.6 nM) (n = 7) channels appeared less sensitive, with the Ca2+ dependence of heteromeric channels being consistent with those that underlie the hippocampal neuron slow afterhyperpolarization. (c) EC50 values for activation of hSK1, hIKCa and hSK1:hIKCa channels by Ca2+. There was no significant difference between EC50 values of hSK1 and hIKCa (p = 0.129) or hSK1 and hSK1:hIKCa channels (p = 0.359). However, there was a significant rightward shift of the EC50 value of hSK1:hIKCa channels compared with hIKCa channels (p = 0.0004***). Significance determined by one‐way ANOVA.

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  FIGURE 9. Apamin binding pocket is absent in heteromeric hSK1:hIKCa channels, while inclusion of the hSK1[S348T + A371V] subunit in heteromeric channels recreates the TRAM‐34 binding domain. (ai) Prediction of hSK1 channel structure by AlphaFold shows the donation of the S3–S4 extracellular loop (yellow) to the adjacent subunit, creating the two‐site binding motif for apamin (between loop [yellow] and outer pore [red]). (aii) Preferential formation of heteromeric channels produces a tetramer where subunits alternate in identity (hSK1, blue; hIKCa, orange). This arrangement of subunits places too great a distance between the S3–S4 extracellular loop and outer pore of the hSK1 subunits within the heteromeric tetramer, rendering the heteromeric channel insensitive to apamin. (bi) In wildtype hIKCa channels, T250 and V275 (magenta) form a TRAM‐34 binding pocket below the selectivity filter. (bii) Heteromeric channels preferentially formed upon co‐expression of wildtype hSK1 (blue) and hIKCa (orange) subunits disrupts this binding pocket and renders the heteromeric channel to be less sensitive to inhibition by TRAM‐34. (biii) Inclusion of the mutated hSK1[S348T + A371V] subunits within…