Two distinct effects of PIP2 underlie auxiliary subunit-dependent modulation of Slo1 BK channels

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca(2+)- and voltage-dependent K(+) (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (α) subunits as well as various regulatory auxiliary subunits (β and γ) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP2 on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP2 inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + γ1 complexes. In contrast, PIP2 markedly increased macroscopic currents through Slo1 + β1 and Slo1 + β4 channel complexes and failed to alter macroscopic currents through Slo1 + β2 and Slo1 + β2 Δ2-19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP2 promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of β subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + γ1, PIP2 augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When β1 or β4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP2 is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP2 may exert distinct tissue- and divalent cation-dependent modulatory influences.

Figure 1.

Effects of PIP₂ on Slo1 complexes with different subunit compositions. (A) Illustrative currents through Slo1, Slo1 + β1, Slo1 + β2, Slo1 + β2 Δ2–19, Slo1 + β4, and Slo1 + LRRC26 (γ1). In each panel, currents before (blue) and after (red) the application of 10 µM PIP₂ to the cytoplasmic side recorded without Ca²⁺ are shown. Pulses were applied from 0 mV every 3 s except for Slo1 + β2, which was stimulated every 10 s. For Slo1 + β2, 1-s prepulses to −100 mV preceded depolarization pulses. For Slo1 + LRRC26 (γ1), the holding voltage was −80 mV. (B) Fractional changes in peak outward currents in Slo1, Slo1 + β1, Slo1 + β2, Slo1 + β2 Δ2–19, Slo1 + β4, and Slo1 + LRRC26 (γ1). (C) Normalized conductance (G-V) curves before (blue) and after (red) the application of 10 µM PIP₂ in the channels indicated. The smooth curves are Boltzmann fits to the results with: Slo1, V_0.5 = 154.5 ± 3.1 mV and Q_app = 1.33 ± 0.04 (Control), and 170.1 ± 2.7 mV and 1.18 ± 0.05 (PIP₂); Slo1 + β1, V_0.5 = 167.8 ± 2.0 mV and Q_app = 0.92 ± 0.02 (Control), and 122.3 ± 2.8 mV and 0.95 ± 0.03 (PIP₂); Slo1 + β2 Δ2–19, V_0.5 = 163.9 ± 3.0 mV and Q_app = 0.98 ± 0.02 (Control), and 158.6 ± 2.4 mV and 0.92 ± 0.02 (PIP₂); Slo1 + β4, V_0.5 = 217.2 ± 3.2 mV and Q_app = 0.99 ± 0.03 (Control), and 183.2 ± 4.0 mV and 1.00 ± 0.03 (PIP₂); and Slo1 + LRRC26 (γ1), V_0.5 = 20.9 ± 3.5 mV and Q_app = 1.38 ± 0.06 (Control), and 42.3 ± 1.8 mV and 1.09 ± 0.04 (PIP₂); n = 9–18. Error bars represent mean ± SEM.

Figure 2.

Changes in kinetics of ionic currents by PIP₂. (A) Scaled representative currents through Slo1, Slo1 + β1, Slo1 + β2 Δ2–19, Slo1 + β4, and Slo1 + LRRC26 (γ1) before (blue) and after (red) the application of 10 µM PIP₂. (B) Time constant (τ) of ionic currents at different voltages before (blue) and after (red) the application of 10 µM PIP₂ in the channels indicated. (C) Fractional changes in time constant of ionic currents by 10 µM PIP₂. All results shown were obtained without Ca²⁺; n = 6 to 12. Error bars represent mean ± SEM.

Figure 3.

PIP₂ increases P_o at negative voltages without Ca²⁺. (A) Representative single-channel openings at −120 mV of Slo1 without Ca²⁺ before and after the application of 10 µM PIP₂. In each condition, 25 data traces are shown superimposed. This patch contained ∼350 channels. (B) Representative single-channel openings at −120 mV of Slo1 + β1 without Ca²⁺ before and after the application of 10 µM PIP₂. 60 data traces are shown superimposed, and the patch contained ∼250 channels. (C) Comparison of P_o changes in Slo1 and Slo1 + β1 by 10 µM PIP₂. (D) Fractional changes in P_o by 10 µM PIP₂; n = 7 and 8 for Slo1 and Slo1 + β1, respectively. All results were obtained without Ca²⁺. Error bars represent mean ± SEM.

Figure 4.

Manipulations of divalent cation concentrations alter the direction of the PIP₂ effect in Slo1. (A) G-V curves before (blue) and after PIP₂ addition (red), and the subsequent addition of 10 mM Mg²⁺ (black), from a representative patch expressing divalent cation–insensitive Slo1 D362A:D367A:E399A:Δ894–895 channels. (B) Changes in V_0.5 of Slo1 D362A:D367A:E399A:Δ894–895 by PIP₂ and Mg²⁺. (C) Representative currents (left) and G-V curves from five patches (right) of wild-type Slo1 before (blue) and after (red) the application of 10 µM PIP₂ in the presence of 2 mM Mg²⁺. (D) Representative currents (left) and G-V curves from five patches (right) containing Slo1 D362A:D367A:E399A:Δ894–895 before (blue) and after (red) the application of 10 µM PIP₂ in the presence of 100 µM Ca²⁺. (E) Representative currents (left) and G-V curves from seven patches (right) containing wild-type Slo1 before (blue) and after (red) the application of 10 µM PIP₂ in the presence of 100 µM Ca²⁺. (F and G) Ca²⁺ dependence of V_0.5 before (blue) and after (red) the application of 10 µM PIP₂ (F) and that of ΔV_0.5 (G) by 10 µM PIP₂. (H) Representative currents from Slo1 D362A:D367A:E399A:Δ894–895 + β1 before (blue) and after (red) the application of 10 µM PIP₂ in the presence of 10 mM Mg²⁺. (I) Changes in V_0.5 of Slo1 D362A:D367A:E399A:Δ894–895 + β1 by PIP₂ and Mg²⁺; n = 8. (J) Representative currents (left) and G-V curves from seven patches (right) of wild-type Slo1 + β1 before (blue) and after (red) the application of 10 µM PIP₂ with 100 µM Ca²⁺ inside. (K) Representative currents (left) and G-V curves from six patches (right) containing wild-type Slo1 recorded in the outside-out configuration before (blue) and after (red) the application of 10 µM PIP₂ to the extracellular side without any added Mg²⁺ or Ca²⁺. The V_0.5 values before and after the application of PIP₂ were 151.7 ± 1.7 mV and 137.2 ± 1.3 mV (P = 0.0021; n = 6). Error bars represent mean ± SEM.

Figure 5.

Critical role of the β N terminus in determining ΔV_0.5 by PIP₂. (A) Sequence alignment of β1, β2 Δ2–19, and β2 Δ2–32 N termini. (B) Changes in G-V parameters by PIP₂ in Slo1 complexes with different β subunits. (C) Sequence alignment of β1, β2–32, and β4 N termini. (D) Changes in G-V parameters by PIP₂ in Slo1 + β1 complexes with the β1-to-β2 point mutations indicated. (E) Changes in G-V parameters by PIP₂ in Slo1 + β1 complexes with β1 mutations at position 11. (F) Changes in G-V parameters by PIP₂ in Slo1 + β2 with the β2-to-β1 point mutations indicated. (G) Changes in G-V parameters by PIP₂ in Slo1 + β4 with the β4-to-β1 (top) and β4-to-β2 (bottom) point mutations indicated. In D–F, the gray shaded areas represent the mean ± SEM of ΔV_0.5 by PIP₂ in Slo1 + β1 (left) and Slo1 + β2 Δ2-32 (right). In G, the gray shaded area shows the mean ± SEM of ΔV_0.5 by PIP₂ in Slo1 + β2 Δ2–32. All results were obtained without Ca²⁺. Error bars represent mean ± SEM.

Figure 6.

Roles of ³²⁹RKK³³¹ and the GR domain in modulation of P_o in Slo1 + β1 by PIP₂. (A) Schematic structural organization of a Slo1 subunit without the GR domain (Slo1ΔGR-Kv-minT). In Slo1ΔGR-Kv-minT, the polypeptide is truncated immediately C terminal to the sequence ³²⁹RKK³³¹. The distal red segment represents the amino acids added by Budelli et al. (2013) (GVKESLGGTDV). (B) Representative currents from Slo1ΔGR-Kv-minT + β1 before (blue) and after (red) the application of 10 µM PIP₂. (C) Fractional changes in peak outward currents at different voltages by 10 µM PIP₂ (red). The gray shaded area shows the mean ± SEM results from wild-type Slo1 + β1 for comparison. (D, F, and H) Illustrative currents through Slo1 R329A:K330A:K331A (D), Slo1 R329A:K330A:K331A + β1 (F), and Slo1 R329A:K330A:K331A + β4 (H) before (blue) and after (red) the application of 10 µM PIP₂. (E, G, and I) G-V curves of Slo1 R329A:K330A:K331A (“RKK mutant”), Slo1 R329A:K330A:K331A (“RKK mutant”) + β1, and Slo1 R329A:K330A:K331A (“RKK mutant”) + β4 before (blue circles) and after (red circles) the application of 10 µM PIP₂. For comparison, G-V curves from the respective wild-type Slo1 (E), Slo1 + β1 (G), and Slo1 + β4 (I) before (blue triangles) and after (red triangles) the application of 10 µM PIP₂ are also shown; n = 6–8. All results were obtained without Ca²⁺. Error bars represent mean ± SEM.