Preferential formation of human heteromeric SK2:SK3 channels limits homomeric SK channel assembly and function

Figures

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  Figure 1. Expression of DsRed with hSK2 or hSK3 subunits.A and B, representative images of cells co-expressing DsRed and hSK2 (A) or hSK3 (B) subunits. Fluorescence shows SK channels (green; left) or DsRed (magenta; centre) with DAPI (blue; left and centre). The right-hand images show a composite of the green and magenta channels only. Scale bars represent 10 μm. White lines overlaying the composite images highlight the cell section used for the intensity plots in C. C, intensity plots showing the relative distribution of DsRed with hSK2 (i) and hSK3 (ii) subunits. Plots were calculated along the lines shown from left (L) to right (R). SK channels, small-conductance, calcium-activated potassium channels.

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  Figure 2. Expression of DsRed with hSK2 and hSK3 subunits. Representative images of cells co-expressing DsRed, hSK2, and hSK3 subunits. A and B, fluorescence shows hSK2 (A; green; left), hSK3 (B; green; left), or DsRed (magenta; centre) with DAPI (blue; left and centre). The right-hand images show a composite of the green and magenta channels only. Scale bars represent 10 μm. White lines overlaying the composite images highlight the cell section used for the intensity plots in C. C, intensity plots showing the relative distribution of DsRed with hSK2 (i) and hSK3 (ii) subunits. Plots were calculated along the lines shown from left (L) to right (R). D, homomeric hSK3 currents were larger than those of homomeric hSK2 or during co-expression of both isoforms. Currents were elicited during whole-cell voltage clamp using voltage ramps and measured at −20 mV. Data are presented as mean ± SD. SK channels, small-conductance, calcium-activated potassium channels.

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  Figure 3. Sensitivity of WT SK channels to apamin.A–C, representative traces following expression of hSK2 (A; n = 7), hSK3 (B; n = 7) or hSK2 + hSK3 (C; n = 7) subunits evoked by a voltage ramp from −100 mV to +100 mV (1 s duration). Under all conditions, current was inhibited by incrementally increasing concentrations of apamin. D, concentration–inhibition relationships show hSK2-mediated current to be more sensitive to apamin than hSK3-mediated current. Co-expression of both isoforms produced current with an intermediate sensitivity. SK channels, small-conductance, calcium-activated potassium channels.

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  Figure 4. Sensitivity of WT SK channels to UCL1684.A–C, representative traces following expression of hSK2 (A; n = 6), hSK3 (B; n = 7), or hSK2 + hSK3 (C; n = 9) subunits evoked by a voltage ramp from −100 mV to +100 mV (1 s duration). Under all conditions, current was inhibited by incrementally increasing concentrations of UCL1684. D, concentration–inhibition relationships show hSK2-mediated current to be more sensitive to UCL1684 than hSK3-mediated current. Co-expression of both isoforms produced current with an intermediate sensitivity. SK channels, small-conductance, calcium-activated potassium channels.

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  Figure 5. Whole-cell current amplitudes of channels containing hSK3(H485N) subunits. The outer pore histidine-to-asparagine mutation in hSK3 (H485N) had no effect on current size when expressed as a homomer or with WT-hSK2. Currents were elicited during whole-cell voltage clamp using voltage ramps and measured at −20 mV. Data are presented as mean ± SD.

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  Figure 6. UCL1684 sensitivity of cells expressing hSK3-H485N subunits alone or with WT-hSK2 subunits.A and B, 100 nM UCL1684 did not inhibit hSK3(H485N)-mediated current (A) but maximally inhibited current during co-expression of hSK3(H485N) and WT-hSK2 (B; 80 ± 4%; n = 8). Control traces are shown in black and traces after application of 100 nM UCL1684 are in gray.

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  Figure 7. Apamin sensitivity of cells expressing hSK3(H485N) subunits alone or with WT subunits.A–C, representative recordings following application of apamin to cells expressing hSK3(H485N) only (A; n = 5); WT-hSK2 and hSK3(H485N) (B; n = 7), or WT-hSK3 and hSK3(H485N) (C; n = 7). D, concentration–inhibition relationship for apamin during each expression condition. Expression of hSK3(H485N) alone (▼) produced a current which was insensitive to apamin. Co-expression with WT-hSK2 (○) produced a response which was best fit by a monophasic Hill equation, whilst co-expression with WT-hSK3 (●) produced a response which was best fit by a biphasic Hill equation. E, the probability of occurrence (Pocc) for each possible channel stoichiometry, calculated assuming the likelihood of inclusion of WT-hSK3 (light gray) or hSK3(H485N) (dark gray) subunits was equivalent.