A PI3 kinase inhibitor found to activate bestrophin 3

Abstract

Bestrophin 3 (Best3), a member of bestrophin Cl channel family, is a CaCl(cGMP) channel candidate in vascular smooth muscle cells. The mechanism for its activation remains unclear. In previous studies, we reported that an autoinhibitory domain ((356)IPSFLGS(362)) existed in Best3 C-terminus and when the autoinhibitory domain was mutated, the Best3 channel was dramatically activated. In this study, we further dissected the roles of the C-terminal sequence in Best3 activation. We found that there were eight basic amino acids downstream of the AI domain within the region (384-397), which were also involved in Best3 activation. Mutations of these basic amino acids significantly activated Best3 as a Cl channel. Led by the assumption that the basic amino acids may be involved in the Best3 C-terminal membrane association through binding to membranous phospholipids, we discovered that PI3Kalpha inhibitor IV could strongly activate Best3.

FIGURE 1

Mutations of basic amino acids in C-terminus activated mBest3. (A) Positions of mutated residues (both numbered and underlined) downstream of the autoinhibitory domain (AI) in mBest3 C-terminus. (B) Stimulatory effect of basic amino acid mutations residing from amino acids 384 to 397 on the mBest3 channel activation. Mutants were transiently transfected into HEK cells, which were voltage-clamped with high [Ca]_i included in the pipette. Step voltages in 20-mV increments or ramp voltages were applied between −100 mV and 100 mV with 1-second duration. The Cl currents were recorded in whole-cell configuration. Internal and external solutions with symmetric [Cl^–]were described in the ”Methods.” Average amplitudes of Cl^– currents at the end of the +100-mV trace are shown. All single or combined mutations except V395A significantly activated mBest3 channel compared with wild-type (WT) mBest3 (P < 0.01, n = 4–7). (C–D) Representatives of current traces produced by ₃₉₃RRKR₃₉₇/AAAA mutant in HEK cells stimulated by step (C) or ramp voltages (D).

FIGURE 2

Association between Best3 C-terminus and membrane was disrupted by alkaline and high ionic strength solutions. Wild type of mBest3 C-terminus was transfected into HEK293 cells, which were sonicated 2 days after transfection with different buffers: 0.1 M Na₂CO₃, pH 11.5 (lanes 1 and 2); and 0.15 or 2 M NaCl, pH 7.3 (lanes 3–6). Cellular proteins in soluble (S) and membrane (M) fractions were extracted for Western blotting (see ”Methods” for details). Immunoblots were probed with anti-mBest3 antibody. The bands with molecular mass at approximately 50 kDa were expected mBest3 C-terminal proteins. The bands with smaller molecular masses (approximately 37 kDa) in lanes 3 and 5 (S) were probably the protease-degraded proteins.

FIGURE 3

PI3Kα inhibitor IV slowly activated mBest3 in a representative HEK cell. The transfected cells were stimulated with ramp and step voltage protocols (see ”Methods” and Fig.1 legend). A, Time course of mBest3 activation by PI3Kα inhibitor IV. The inhibitor (10 μM) applied to the bath solution slowly induced Cl currents recorded with ramp voltages. Step voltages were applied twice in the middle (B) and the end (C) of recordings to observe the current wave forms. The induced currents were blocked by SO₄^–2 to eliminate the possibility of leaky currents. Note that the outward currents were completely blocked by extracellular SO₄^–2, whereas inward currents were not. B, First-step voltage stimulation in A in the presence of the inhibitor (B). Currents behaved as outwardly rectifying. C, Step voltage stimulation in A in absence of the inhibitor (C). The currents were time- and voltage-independent when the inhibitor had been washed out. D, Ramp currents that were blocked by SO₄^–2 showed the linear pattern with V_rev close to zero.

FIGURE 4

PI3Kα inhibitor IV and Best3 activation. Average amplitudes of mBest3 currents activated by PI3Kα inhibitor IV. Wild-type mBest3 in pcDNA3.1 vector was cotransfected into HEK cells with GFP or cells were transfected with GFP only. Green fluorescent cells were selected for whole-cell recordings with ramp voltage stimulations (refer to Fig.1 legend). The PI3K inhibitor (10 μM) applied to the bath dramatically stimulated Cl currents from mBest3-transfected (n = 7) but not from GFP-only transfected HEK cells (n = 4). The currents obtained with 100-mV stimulation, 10 minutes after the PI3K inhibitor application to two groups, were averaged for statistical analysis (P < 0.01).