Modulation of BK Channel Gating by the β2 Subunit Involves Both Membrane-Spanning and Cytoplasmic Domains of Slo1

Differential modulation by β2ND on Ca²⁺ sensitivity of mSlo1 and dSlo1. A, Macroscopic currents of mSlo1 and dSlo1 with WT hβ2 at ∼100 μm [Ca²⁺]_i (left). The voltage pulses are from −100 to +150 mV for 1 s with 25 mV increments (shown only the first 20 ms), and the prepulse potential is −140 mV for 195 ms (shown only the last 5 ms). The dotted line represents the biexponential fit of the inactivation profile, and the long dashed line represents the zero current line for each current trace. On the right, voltage dependence of the inactivation time constant is shown. The fast and slow components of τ_Inact were obtained by fitting current traces with a biexponential function from the peak amplitude to steady state (n = 10 for mSlo1 + WT β2, except at +150 mV, n = 9; n = 5 for dSlo1 + WT β2, except at +150 mV, n = 4). B, Macroscopic currents of mSlo1 and dSlo1 with and without β2ND at ∼100 μm [Ca²⁺]_i. Voltage pulses are from −200 to 100 mV with 10 mV increments, and the repolarizing potential is −50 mV, except for mSlo1 + β2ND, which is −80 mV. The dashed line represents the zero current line for each current trace. C, Mean G–V relationship of mSlo1/dSlo1 with and without β2ND in 0, ∼2, and ∼100 μm [Ca²⁺]_i, fitted with Boltzmann equation (smooth lines). The error bars in this and other figures show the SEM. D, V_½ versus [Ca²⁺]_i plot of mSlo1/dSlo1 with and without β2ND. The number of patches for each dataset are for the following (μm): [Ca²⁺]_i, 0, 1, 2, 5, 10, 30, 100; mSlo1, 118, 33, 29, 29, 42, 25, 56; mSlo1 + β2ND, 44, 8, 8, 11, 9, 12, 28; dSlo1, 0, 0, 11, 19, 15, 9, 27; dSlo1 + β2ND, 0, 0, 3, 4, 7, 8, 14.

Effects of chimeras of mSlo1/dSlo1 on the β2ND modulation of Ca²⁺ dependence. The black bars show the change in Ca²⁺ sensitivity between with and without β2ND, normalized to mSlo1 (see Results for definitions). The gray bars show the Ca²⁺ response at ∼100 μm [Ca²⁺]_i between with and without β2ND. The symbol * means that ∼2 μm [Ca²⁺]_i was used for comparison, and ** means that the G–V could not be determined at 0 or ∼2 μm [Ca²⁺]_i. Bottom shows the schematic of different chimeras of mSlo1 and dSlo1 used in the top. The number of patches for each dataset are as follows for either ∼0/2 and 100 μm [Ca²⁺]_i: C1, 3, 5; C1 + β2ND, 9, 11; C2, 4, 3; C2 + β2ND, 5, 4; C3, 7, 4; C3 + β2ND, 5, 5; C4, 5, 5; C4 + β2ND, 8, 3; C5, 7, 5; C5 + β2ND, 3, 4; m[dS0]: 9, 6; m[dS0] + β2ND, 4, 7; m[dLinker + dAC], 7, 20; m[dLinker + dAC] + β2ND, 0, 20; m[dS0 + dLinker + dAC], 5, 14; m[dS0 + dLinker + dAC] + β2ND: 2, 9; d[mS0], 3, 20; d]mS0] + β2ND, 0,12; d[mLinker + mAC], 13, 7; d[mLinker + mAC] + β2ND, 23, 13; d[mS0 + mLinker + mRCK1c], 11, 16; d[mS0 + mLinker + mRCK1c] + β2ND, 15, 14.

Effects of the N terminus and the C-Linker/AC region of dSlo1 in mSlo1 background on the β2ND modulation of Ca²⁺ sensitivity. A, Macroscopic currents of m[dS0], m[dLinker + dAC], and m[dS0 + dLinker + dAC] with and without β2ND at ∼100 μm [Ca²⁺]_i. Voltage pulses are from −200 to 200 mV with 10 mV increments, and the repolarizing potential is −50 mV, except for m[dS0] and m[dLinker + dAC] with β2ND, which is −80 mV. The dashed line represents the zero current line for each current trace. B, G–V relations of chimeras compared with that of mSlo1 (orange) at low (0, ∼2, or ∼10 μm) and ∼100 μm [Ca²⁺]_i. Because of the apparent inactivation of the channels, the G–V relations were measured from both the peak currents and tail currents, and both methods resulted in the same V_½. C, V_½ versus [Ca²⁺]_i plots of chimeras compared with that of mSlo1 and dSlo1 with and without β2ND. The number of patches for each dataset are as follows (in μm): [Ca²⁺]_i, 0, 1, 2, 5, 10, 30, 100; m[dS0], 9, 6, 4, 4, 4, 4, 6; m[dS0] + β2ND, 4, 3, 3, 4, 3, 5, 7; m[dLinker + dAC], 0, 0, 7, 14, 15, 6, 20; m[dLinker + dAC] + β2ND, 0, 0, 0, 0, 4, 5, 20; m[dS0 + dLinker + dAC], 0, 0, 5, 4, 4, 4, 14; m[dS0 + dLinker + dAC] + β2ND, 0, 0, 2, 3, 4, 2, 9.

Effects of the N terminus and C-Linker/AC region of mSlo1 in dSlo1 background on the β2ND modulation of Ca²⁺ sensitivity. A, Macroscopic currents of d[mS0], d[mLinker + mAC], and d[mS0 + mLinker + mRCK1c] with and without β2ND at ∼100 μm [Ca²⁺]_i. Voltage pulses are from −200 to 200 mV, except for d[mS0 + mLinker + mRCK1c] + β2ND, which are from −200 to 100 mV, with 10 mV increments. The repolarizing potential is −50 mV. The dashed line represents the zero current line for each current trace. B, G–V relations of chimeras with and without β2ND at low (0 or ∼2 μm) and ∼100 μm [Ca²⁺]_i compared with that of dSlo1 (blue). C, V_½ versus [Ca²⁺]_i plots of chimeras compared with that of mSlo1 and dSlo1 with and without β2ND. The number of patches for each dataset are as follows (in μm): [Ca²⁺]_i, 0, 1, 2, 5, 10, 30, 100; d[mS0], 0, 0, 3, 10, 6, 4, 20; d[mS0] + β2ND, 0, 0, 0, 3, 2, 3, 16; d[mLinker + mAC], 13, 5, 4, 4, 4, 4, 7; d[mLinker + mAC] + β2ND, 23, 5, 3, 3, 5, 5, 13; d[mS0 + mLinker + mRCK1c], 11, 2, 4, 7, 6, 16; d[mS0 + mLinker + mRCK1c] + β2ND, 15, 5, 5, 5, 6, 14.

MWC model fittings of WT and chimera channels. G–V relationships (symbols) for mSlo1, dSlo1, m[dS0 + dLinker + dAC], and d[mS0 + mLinker + mRCK1c] with and without β2ND fitted with the MWC model (lines). The [Ca²⁺]_i for each symbol is shown.

The role of the C-Linker and AC region of mSlo1 in the β1 and β2ND modulation of Ca²⁺ sensitivity. A, G–V relations of mSlo1 and m[dLinker + dAC] with and without β1 or β2ND in ∼100 μm [Ca²⁺]_i. B, V_½ versus [Ca²⁺]_i plots of mSlo1 and m[dLinker + dAC] with β1 (left) or β2ND (right). The number of patches are as follows (in μm): [Ca²⁺]_i, 0, 1, 2, 5, 10, 30, 100: mSlo1 + β1, 38, 13, 15, 11, 12, 8, 21; m[dLinker + dAC] + β1, 0, 0, 0, 10, 6, 9, 26.

Dependence of the β1 and β2 modulation of Ca²⁺ sensitivity on Ca²⁺ binding sites in mSlo1. A, Bar graph of the G–V shift in response to a [Ca²⁺]_i change from 0–100 μm WT, single Ca²⁺ binding site mutations (D367A and 5D5N), double Ca²⁺ binding site mutation (D367A/5D5N), and double Ca²⁺ binding site plus the Mg²⁺ binding site mutation (D367A/5D5N/E399N) without (black) and with β1 (white) or β2ND (gray). B, Differences in ΔV_½ from A of β1 (white) and β2ND (gray) with WT, single, double, and triple binding site mutations. The number of patches for each dataset are as follows for ∼0 and ∼100 μm [Ca²⁺]_I, respectively: mD367A, 10, 9; mD367A + β1, 9, 9; mD367A + β2ND, 5, 7; 5D5N, 6, 11; 5D5N + β1, 8, 9; 5D5N + β2ND, 4, 8; mD367A/5D5N, 14, 6; mD367A/5D5N + β1, 6, 5; mD367A/5D5N + β2ND, 5, 5; mD367A/5D5N/E399N, 15, 15; mD367A/5D5N/E399N + β1, 5, 9; mD367A/5D5N/E399N + β2ND, 8, 12.