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The protein concentration dependence of molar masses of free SKp and CaM determined from the self+hetero-association protocol measured at high Ca2+ (closed circles) or zero Ca2+ (open circles). Data for SKp are black symbols; CaM data are shown with orange symbols.
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Determination of the molar mass by MALS. (A and B) Data for SEC-MALS. The horizontal broken lines are the predicted molar masses given by the left vertical axes for free CaM (orange broken lines), a 1/1 SKp/CaM complex (blue broken lines), and a 2/2 complex (lavender broken lines). The solid traces are the change in the differential refractive index (ΔRI scale on right vertical axis) after size-exclusion chromatography fractionation as determined by an in-line refractometer. The closed circles show the molar mass calculated from MALS. (A) 100 µM free CaM (orange data) or 100 µM molar mixture of CaM + SKp (blue data) was fractionated in a low-salt solution of 100 mM NaCl, 5 mM HEPES, and 1 mM CaCl2, followed by MALS. (B) The red trace shows the elution profile of the 29 kD complex (peak 1) formed as 100 µM CaM in a buffer of 500 mM NaCl, 5 mM HEPES, and 1 mM CaCl2, pH 7.0, which binds to and releases previously bound SKp from the column. Peak 2 represents free CaM. The black broken ΔRI curve and the black open circles are the same data from A for the SKp/CaM complex in low salt.
A faster sedimentation indicates a larger mass, a change in the hydrodynamic volume, or a combination of these two factors. A different stoichiometry will have a different mass. Thus further analyses of the sedimentation velocity data are needed to determine whether excess of either SKp or CaM alters either the stoichiometry or the hydrodynamic volume of the complex. Because vHW is better suited for heterogeneous experiments, dC/dt is not used for molar ratios with more than twofold differences in concentrations of SKp to CaM. When SKp is at least fivefold molar excess over CaM, the absorbance profile of the scans show two different plateau phases (Fig. 7). Applying vHW analysis clearly resolves the sedimentation coefficients of both species. Instead of vertical, the profile has one phase that aligns with the 2SKp:1CaM data and another phase that aligns with the free SKp data (Fig. 9 C). The simplest interpretation is that these phases align with a 2SKp/1CaM complex and free SKp. Similarly, when CaM is in 10-fold molar excess over SKp, vHW analysis resolves a phase that aligns with the 1SKp:2CaM data and a second phase that aligns with free CaM (Fig. 9 D). In this case, we resolve the 1SKp/2CaM and free CaM. Together, these data support our interpretations of the CG-MALS data where both 2SKp/1CaM and 1SKp/2CaM stoichiometries were observed at saturating Ca2+.
The 2/2 gating model proposes a modular role of CaM in SK activation. At low Ca2+, the complex that forms is 1SK/1CaM, with only the C-lobe of CaM engaged. An increase in intracellular Ca2+ drives Ca2+ binding only to the N-lobe of CaM, and a 2SK/2CaM interaction forms. Although the 2/2 gating model is consistent with the crystallographic results for a single conformational state, there has been some physiological evidence that it is insufficient to describe channel gating. For instance, the part of CaM responsible for Ca2+ dependent binding, the N-terminal lobe, also has an effect on Ca2+-free interactions with SK (Li et al., 2009). It is clear that our understanding of the role of CaM in SK gating is inadequate.
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