## Results: 12

Figure 5. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

^{2+}sensitivity between mSlo1 and dSlo1. Plot of (A) V

_{1/2}and (B) z versus [Ca

^{2+}]

_{i}for mSlo1, dSlo1, m[dAC], and d[mAC]. V

_{1/2}and z are obtained by fitting the G-V relations of the channels at various [Ca

^{2+}]

_{i}with the Boltzmann equation.

Figure 11. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

^{2+}binding to closed channels. (A and B) G-V relations of mSlo1, dSlo1, d[mAC], and m[dAC] channels at [Ca

^{2+}]

_{i}of 0, 1.7, 2.3, 5.7, 11.2, 28.5, and 89 μM. Each dataset was fit (smooth curves) by the MWC model (),

*n*= 4 (A) or 8 (B). (C) Parameters of fits obtained from A and B.

Figure 12. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

^{2+}and in the closed conformation, the AC region adopts a conformation that inhibits channel opening. Ca

^{2+}binding (+Ca

^{2+}) or channel opening by depolarization (+V) removes this inhibition, rendering the channel gate more favorable to the open conformation. In the open conformation or when the Ca

^{2+}binding sites are occupied, the AC region has little effect on channel gating.

Figure 3. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

^{2+}]

_{i}. (A and B) Current traces of dSlo1 for indicated [Ca

^{2+}]

_{i}elicited by voltages from −150 to 200 mV. (C) Steady-state G-V curves of dSlo1. Solid curves are fits of the Boltzmann equation. V

_{1/2}obtained from the fits of the G-V relations at each [Ca

^{2+}]

_{i}is indicated. Dotted lines indicate MWC model simulations of the G-V relation at [Ca

^{2+}]

_{i}89, 100, 200, and 300 μM using the same parameters as indicated in C.

Figure 2. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

^{2+}]

_{i}of 2, 10, and 100 μM. Test potentials were −80 to +200 mV, holding and repolarization potentials were −50 mV. (Bottom) Steady-state G-V relations of Chim2 and mSlo1. Smooth curves are fits of the Boltzmann equation with parameters for Chim2, in 2 μM [Ca

^{2+}]

_{i}: V

_{1/2}= 240.1 mV, z = 0.95, in 10 μM [Ca

^{2+}]

_{i}: V

_{1/2}= 76.1 mV, z = 1.5, in 100 μM [Ca

^{2+}]

_{i}: V

_{1/2}= 43.5 mV, z = 1.5; for mSlo1, in 2 μM [Ca

^{2+}]

_{i}: V

_{1/2}= 84.8 mV, z = 1.5, in 10 μM [Ca

^{2+}]

_{i}: V

_{1/2}= 31.4 mV, z = 1.5, in 100 μM [Ca

^{2+}]

_{i}: V

_{1/2}= 14.4 mV, z = 1.4. (B, top) Current traces of Chim6 and mSlo1 at [Ca

^{2+}]

_{i}of 0, 10, and 500 μM. Test potentials were −80 to +200 mV, holding and repolarization potentials were −50 mV. (Bottom) Steady-state G-V relations of Chim6 and mSlo1. Smooth curves are fits of the Boltzmann equation with parameters for Chim6, in 0 [Ca

^{2+}]

_{i}: V

_{1/2}= 206.7 mV, z = 1.3, in 10 μM [Ca

^{2+}]

_{i}: V

_{1/2}= 101.1 mV, z = 1.2, in 500 μM [Ca

^{2+}]

_{i}: V

_{1/2}= 52.5 mV, z = 1.2; for mSlo1, in 0 [Ca

^{2+}]

_{i}: V

_{1/2}= 189.7 mV, z = 1.1, in 10 μM [Ca

^{2+}]

_{i}: V

_{1/2}= 33.8 mV, z = 1.7, in 500 μM [Ca

^{2+}]

_{i}: V

_{1/2}= −13.2 mV, z = 1.8.

Figure 9. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

_{α}'s of the above structure. The purple box denotes the same region (αB) highlighted above. Amino acid in the sequence at the bottom corresponds to the C

_{α}whose dynamics are plotted above. Molecular graphics were produced using visual molecular dynamics (VMD) ().

Figure 7. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

^{2+}sensitivity of BK

_{Ca}gating. (A) Steady-state G-V relations of m[d-Motif2] and m[d-Motif3]. Dotted lines are G-V relations of mSlo1. Parameters of the Boltzmann fits (solid lines): for m[d-Motif2], at 0 [Ca

^{2+}]

_{i}, V

_{1/2}= 195.7 mV, z = 1.1; at 100 μM [Ca

^{2+}]

_{i}, V

_{1/2}= 8.2 mV, z = 1.2; for m[d-Motif3], at 0 [Ca

^{2+}]

_{i}, V

_{1/2}= 213.2 mV, z = 1.1; at 100 μM [Ca

^{2+}]

_{i}, V

_{1/2}= 9.2 mV, z = 1.2. (B) Steady-state G-V relations of m[d-Motif12] and m[d-Motif13]. Dotted lines are G-V relations of mSlo1. Parameters of the Boltzmann fits (solid lines): for m[d-Motif12], at 0 [Ca

^{2+}]

_{i}, V

_{1/2}= 186.5 mV, z = 1.2; at 100 μM [Ca

^{2+}]

_{i}, V

_{1/2}= 22.2 mV, z = 1.1; for m[d-Motif13], at 0 [Ca

^{2+}]

_{i}, V

_{1/2}= 211.9 mV, z = 1.4; at 100 μM [Ca

^{2+}]

_{i}, V

_{1/2}= 38.4 mV, z = 1.3. (C) Free energy of activation provided by Ca

^{2+}binding for the chimeric channels. Free energy change for each channel was in response to [Ca

^{2+}]

_{i}change shown in parentheses for mSlo1 plotted alongside each group.

Figure 1. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

^{2+}sensitivity than mSlo1. (A) Current traces of mSlo1 and dSlo1. Test potentials were −80 to +200 mV and −150 to +200 mV for 5.7 μM and 89 μM [Ca

^{2+}]

_{i}, respectively, holding and repolarization potentials were −50 mV. (B) Steady-state G-V relations of mSlo1 and dSlo1. Smooth curves are fits of the Boltzmann equation (see MATERIALS AND METHODS) with parameters for mSlo1, in 0 [Ca

^{2+}]

_{i}: V

_{1/2}= 179 mV, z = 1.2, in 5.7 μM [Ca

^{2+}]

_{i}, V

_{1/2}= 64.6 mV, z = 1.4, in 89 μM [Ca

^{2+}]

_{i}, V

_{1/2}= −2.8 mV, z = 1.2; for dSlo1, in 5.7 μM [Ca

^{2+}]

_{i}, V

_{1/2}= 157 mV, z = 1.3, in 89 μM [Ca

^{2+}]

_{i}, V

_{1/2}= −2.9 mV, z = 1.1. (C) Free energy provided by Ca

^{2+}binding for channel activation when [Ca

^{2+}]

_{i}changes from 0 or 5.7 μM to 89 μM, as indicated in parentheses under the abscissa. m, mSlo1; d, dSlo1.

Figure 4. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

^{2+}sensitivity. (A) Ca

^{2+}sensitivity of activation in chimeric channels of mSlo1 and dSlo1. The vertical axis shows schematic representation of chimera constructs with dSlo1 portions shaded gray and mSlo1 black. Rectangles are transmembrane segments or RCK1 domains (), ovals are the Ca

^{2+}bowl (). Free energy increase in response to increase in [Ca

^{2+}]

_{i}(shown at the right) for each chimera and WT channel was normalized against that for mSlo1. (B) Plot of V

_{1/2}versus [Ca

^{2+}]

_{i}for mSlo1 (thin black line), dSlo1 (thick black line), and the chimeric channels as defined in A. V

_{1/2}values are obtained by fitting the G-V relations of the channels at various [Ca

^{2+}]

_{i}with the Boltzmann equation.

Figure 8. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

^{2+}sensitivity in dSlo1 is not related to the low affinity metal binding site. (A) Steady-state G-V relations of mSlo1 and dSlo1 at 89 μM [Ca

^{2+}]

_{i}and indicated [Mg

^{2+}]

_{i}. Boltzmann fits (smooth curves) gave following parameters: mSlo1, for 0 [Mg

^{2+}]

_{i}, V

_{1/2}= −2.8 mV, z = 1.2, for 10 mM [Mg

^{2+}]

_{i}, V

_{1/2}= −73.8 mV, z = 1.1; dSlo1, for 0 [Mg

^{2+}]

_{i}, V

_{1/2}= −2.9 mV, z = 1.1, for 10 mM [Mg

^{2+}]

_{i}, V

_{1/2}= −69.2 mV, z = 0.94. (B) Steady-state G-V relations of the E413R mutant dSlo1 channel. Boltzmann fits (solid curves) gave the following: E413R, for 5.7 μM [Ca

^{2+}]

_{i}, V

_{1/2}= 163 mV, z = 1.1, for 89 μM [Ca

^{2+}]

_{i}, 0 [Mg

^{2+}]

_{i}, V

_{1/2}= −24.6 mV, z = 0.95, for 89 μM [Ca

^{2+}]

_{i}, 10 mM [Mg

^{2+}]

_{i}, V

_{1/2}= −46.7 mV, z = 0.87. Dotted lines are G-V relations for dSlo1 at 5.7 μM [Ca

^{2+}]

_{i}and 89 μM [Ca

^{2+}]

_{i}, 0 [Mg

^{2+}]

_{i}. (C) Free energy provided by Mg

^{2+}binding for channel activation in mSlo1, dSlo1, and E413R dSlo1 when [Mg

^{2+}]

_{i}changes from 0 to 10 mM, measured at [Ca

^{2+}]

_{i}of 89 μM. (D) Free energy provided by Ca

^{2+}binding for channel activation in WT and E413R dSlo1 when [Ca

^{2+}]

_{i}changes from 5.7 to 89 μM.

Figure 10. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

_{Ca}activation by the AC region depends on Ca

^{2+}occupancy. (A) Current traces of mSlo1, dSlo1, d[mAC], and m[dAC] at 0 [Ca

^{2+}]

_{i}. Test voltages were from −80 to 200 mV with a holding and repolarization potential of −50 mV. (B) Steady-state G-V relations of above channels at 0 (open symbols) and 89 μM (filled symbols) [Ca

^{2+}]

_{i}. Smooth curves are fits to the Boltzmann equation. At 89 μM [Ca

^{2+}]

_{i}, for mSlo1: V

_{1/2}= −2.8 mV, z = 1.2; for d[mAC]: V

_{1/2}= 7.7 mV, z = 1.4; for dSlo1: V

_{1/2}= −2.9 mV, z = 1.1; for m[dAC]: V

_{1/2}= 41.6 mV, z = 1.2. At 0 [Ca

^{2+}]

_{i}, for mSlo1: V

_{1/2}= 179 mV, z = 1.2; for d[mAC]: V

_{1/2}= 181 mV, z = 1.3. The voltage range of dSlo1 and m[dAC] activation at 0 [Ca

^{2+}]

_{i}is too positive to record any current. (C) Box plot of ΔG

_{V}= zV

_{1/2}for the above channels at 0 and 89 μM [Ca

^{2+}]

_{i}. The percentile values shown are 10, 25, 50, 75, and 90 for each channel. ΔG

_{V}for m[dAC] and dSlo1 at 0 [Ca

^{2+}]

_{i}are too large to be determined.

Figure 6. From: The NH2 Terminus of RCK1 Domain Regulates Ca2+-dependent BKCa Channel Gating.

^{2+}sensitivity change is not related to the Ca

^{2+}binding site in the AC region. (A) Sequence alignment of the AC region of the RCK1 domain () from mSlo1 (), dSlo1 (), and the

*archeon*MthK (). Numbers indicate the position of the rightmost residues in the primary sequence of respective proteins. Secondary structures βA-C and αA-C are indicated by underlines. Boxed amino acids labeled as motifs 1, 2, and 3 are regions showing significant sequence differences between mSlo1 and dSlo1. Motif 1 is important for Ca

^{2+}-dependent activation (; ). Effects of switching motif 1 between mSlo1 and dSlo1 are shown in B and C. (B, left) Steady-state G-V relations of the mutant channel m[d1] (motif 1 from dSlo1 in the mSlo1 background). Solid lines are fits of the Boltzmann equation with the following parameters: at 0 [Ca

^{2+}]

_{i}, V

_{1/2}= 185 mV, z = 1.2; at 89 μM [Ca

^{2+}]

_{i}, V

_{1/2}= 15.9 mV, and z = 1.2. Dotted lines are G-V relations of mSlo1. (B, right) Steady-state G-V relations of the mutant channel d[m1] (motif 1 from mSlo1 in the dSlo1 background). Solid lines are fits of the Boltzmann equation with the following parameters: at 89 μM [Ca

^{2+}]

_{i}, V

_{1/2}= −52.5 mV, z = 0.83; at 5.7 μM [Ca

^{2+}]

_{i}, V

_{1/2}= 95.1 mV and z = 0.89; at 0 [Ca

^{2+}]

_{i}, the G-V relation was too right shifted for z and V

_{1/2}values to be determined. Dotted lines are G-V relations of dSlo1. (C) Free energy of activation provided by Ca

^{2+}binding in mSlo1, d[m1], m[d1], and dSlo1 when [Ca

^{2+}]

_{i}increased from 0 to 89 μM (for m[d1]) and from 5.7 to 89 μM (for d[m1] and dSlo1), normalized against corresponding free energy values for mSlo1.