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Small conductance calcium-activated potassium (SK) channels respond to intracellular Ca(2+) via constitutively associated calmodulin (CaM). Previous studies have proposed a modular design for the interaction between CaM and SK channels. The C-lobe and the linker of CaM are thought to regulate the constitutive binding, whereas the N-lobe binds Ca(2+) and gates SK channels. However, we found that coexpression of mutant CaM (E/Q) where the N-lobe has only one functional EF hand leads to rapid rundown of SK channel activity, which can be recovered with exogenously applied wild-type (WT), but not mutant, CaM. Our results suggest that the mutation at the N-lobe EF hand disrupts the stable interaction between CaM and SK channel subunits, such that mutant CaM dissociates from the channel complex when the inside of the membrane is exposed to CaM-free solution. The disruption of the stable interaction does not directly result from the loss of Ca(2+)-binding capacity because SK channels and WT CaM can stably interact in the absence of Ca(2+). These findings question a previous conclusion that CaM where the N-lobe has only one functional EF hand can stably support the gating of SK channels. They cannot be explained by the current model of modular interaction between CaM and SK channels, and they imply a role for N-lobe EF hand residues in binding to the channel subunits. Additionally, we found that a potent enhancer for SK channels, 3-oxime-6,7-dichloro-1H-indole-2,3-dione (NS309), enables the recovery of channel activity with CaM (E/Q), suggesting that NS309 stabilizes the interaction between CaM and SK channels. CaM (E/Q) can regulate Ca(2+)-dependent gating of SK channels in the presence of NS309, but with a lower apparent Ca(2+) affinity than WT CaM.
Figure 1. Coexpression of CaM (E/Q) leads to quick rundown of SK current. (A) Representative current traces recorded under voltage clamp with an inside-out patch excised from a Xenopus oocyte injected with RNA for SK channels and CaM (E1Q). Voltage protocol is shown on top. For clarity, only one trace recorded at the beginning of each minute after patch excision is shown. Horizontal dashed lines in this and other figures represent zero current level. (B) Average current level at â80 mV measured every 3 s from the same patch in A is plotted as a function of time. The jump in current at the beginning of the time course in this and later figures indicates patch excision. Data points during solution change were noisy and removed from the plot in this and other figures. Solid line represents a single-exponential fit of the time course with Ï = 108 s. (C) Representative current traces recorded and shown as in A with a patch excised from an oocyte injected with RNA for CaM (WT) and SK channels. Note a small shift in the reversible potential during the course of recording. (D) Average current level at â80 mV from the patch shown in C measured and plotted as in B.
Figure 2. CaM (WT) can recover SK channel activity. (A) Representative current traces with a patch from a CaM (E2Q)âcoinjected oocyte at the following time points: (1) immediately after patch excision; (2) after rundown stabilized; (3) 50 min after the application of 20 µM CaM (WT); (4) the application of solution containing 16 µM Ba2+; and (5) finally in Ca2+-free solution. (B) Average current level at â80 mV measured every 3 s with the patch shown in A during the course of rundown and recovery. Legends in this and later figures show the different solutions that the patch is subjected to. Solid line represents fit with a single-exponential time course (Ï = 39.7 min). (C) With the same patch shown in A and B, after recovery of SK channel activity with CaM (WT), solutions containing different free Ca2+ concentrations were applied to the patch. Average current level at â80 mV was measured at each concentration, normalized to the maximal value, and plotted as a function of Ca2+ concentration. The doseâresponse relationship is fitted with the Hill equation: I/Imax = 1/(1+(EC50/[ Ca2+])h), yielding EC50 = 0.94 µM and h = 2.6. (D) A representative time course of the average current level at â80 mV with a patch excised from an oocyte coinjected with RNA for SK channels and CaM (WT).
Figure 3. SK channels recovered with CaM (T80D) have reduced apparent Ca2+ affinity. (A) Representative doseâresponse relationships for SK channels recovered with CaM (WT) (filled circles), CaM (T80A) (open squares), and CaM (T80D) (filled triangles). In all cases, patches were excised from oocytes coinjected with RNA for SK channels and CaM (E/Q). Currents were allowed to run down before the application of 20 µM of individual types of CaM. Doseâresponse relationships were measured in CaM-free solutions. Solid lines are fits with the Hill equation (WT, EC50 = 0.97 µM and h = 3.8; T80A, EC50 = 1.03 µM and h = 3.0; T80D, EC50 = 1.82 µM and h = 3.4). (B) Representative doseâresponse relationships for SK channels coexpressed with CaM (WT) (filled circles), CaM (T80A) (open squares), or CaM (T80D) (filled triangles). Solid lines are fits with the Hill equation (WT, EC50 = 1.03 µM and h = 3.7; T80A, EC50 = 1.14 µM and h = 3.1; T80D, EC50 = 1.93 µM and h = 3.3). (C) Average values for EC50 from experiments as shown in A and B. Doseâresponse relationships were individually fitted for each experiment. Error bars represent standard deviation. Trial numbers are shown in the brackets on the top. Labeling is interpreted as the following: SK only-inj, oocytes were injected with SK RNA alone; WT-coinj, CaM (WT) RNA was coinjected with SK RNA; WT-rec, SK channels recovered by CaM (WT) in patches from oocytes coinjected with RNA for the SK channel and CaM (E/Q). The same applies for CaM (T80A) and (T80D). EC50 values for T80D-rec and T80D-coinj are not significantly different from each other (P > 0.05; Student's t test), but significantly different from all other columns (P < 0.01), and are therefore labeled with stars. All other columns are not significantly different from each other (P > 0.05).
Figure 4. CaM (E/Q) can only recover SK channel activity in the presence of NS309. (A) Time course of the average SK current level at â80 mV with a patch excised from an oocyte coexpressing SK channels and CaM (E2Q). Patch was subjected to solutions containing different amount of Ca2+ and 20 µM WT or mutant CaM as shown by the legend. (B) Time course of the average SK current level at â80 mV with a patch from an oocyte coexpressing SK channels and CaM (E1Q). (C) A representative doseâresponse relationship for SK channels recovered with CaM (E2Q) and NS309 in a patch from an oocyte coexpressing SK channels and CaM (E2Q) (open circles). Initial currents were allowed to run down before the application of 20 µM CaM (E2Q) and 100 µM NS309. After recovery, the doseâresponse relationship was measured in the presence of 20 µM CaM (E2Q) and 100 µM NS309. Dashed line represents a fit of the data with a single Hill equation (EC50 = 0.41 µM and h = 1.0). Solid line represents a fit of the data with a two-component Hill equation, I/Imax = c/(1+(EC50a/[ Ca2+])ha) + (1âc)/(1+(EC50b/[Ca2+])hb), in which c is the fraction of the first component (c = 0.41, EC50a = 90 nM, and ha = 2.9; EC50b = 1.14 µM and hb = 1.9). Filled circles are a representative doseâresponse relationship in the presence of 100 µM NS309 for CaM (WT)ârecovered SK channels. Solid line is a fit with a single Hill equation (EC50 = 69 nM and h = 4.2). (D) A representative experiment where NS309 was applied in the absence of CaM to a patch from an oocyte coexpressing SK channels with CaM (E/Q). (E) An example of recovery using CaM (E1Q) and NS309 without pretreatment with CaM (E1Q) alone.
Figure 5. Recovery of SK channels in the absence of Ca2+. (A) Time course of the average current level at â80 mV with a patch from an oocyte coexpressing SK channels with CaM (E1Q). Solid line is a single-exponential fit with a Ï = 39.1 min. (B) After patch was excised from an oocyte expressing SK channel and CaM (E1Q), current ran down to 60 pA before the patch was subjected to 20 µM CaM (WT) in Ca2+-free solution for 60 min. Then, recording pipette was quickly moved into a laminar flow of solution containing 7.7 µM Ca2+ to measure the time course of activation. Current was continuously recorded at â80 mV during the movement and shown in the top panel. Later in the experiment, pipette was moved back to Ca2+-free solution and then to 7.7 µM Ca2+ again (middle). Solid lines are fits of data with a single-exponential time course (top, Ï = 46 ms; middle, Ï = 38 ms). The bottom panel compares the time constants for the first activation after recovery to the average values for later activations. Results were from six similar experiments. (C) Similar experiment as in B, except that CaM (WT) was replaced by 20 µM CaM (E1Q) plus 100 µM NS309, which were also present during the measurement of the activation time course. Before the application of Ca2+-free solution containing CaM (E1Q) and NS309, there was 18 pA of residual current after rundown. Solid lines are fits of data with a single-exponential time course (top, Ï = 70 ms; middle, Ï = 66 ms). Data from five similar experiments are shown in the bottom panel as in B.
Figure 6. CaM (E34Q), but not CaM (E12Q), can recover SK channel activity. (A) Time course of the average current level at â80 mV with a patch from an oocyte coexpressing SK channels with CaM (E2Q) while the patch was subjected to different solutions as indicated by the legend. (B) Time course of the average current level at â80 mV with a patch from an oocyte coexpressing SK channels with CaM (E2Q). (C) Doseâresponse relationship for the patch shown in B was measured after recovery of SK channels with CaM (E34Q) (open circles). Solid line is a fit with the Hill equation (EC50 = 1.24 µM and h = 2.7). Filled circles represent the doseâresponse relationship measured with a patch from an oocyte coexpressing SK channels with CaM (E34Q) fitted with the Hill equation (EC50 = 1.20 µM and h = 3.7).
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