Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Abstract

Plasma membrane large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK(Ca) channel regulation by IP(3) and IP(3)Rs in rat and mouse cerebral artery smooth muscle cells. IP(3) activated BK(Ca) channels both in intact cells and in excised inside-out membrane patches. IP(3) caused concentration-dependent BK(Ca) channel activation with an apparent dissociation constant (K(d)) of approximately 4 microM at physiological voltage (-40 mV) and intracellular Ca(2+) concentration ([Ca(2+)](i); 10 microM). IP(3) also caused a leftward-shift in BK(Ca) channel apparent Ca(2+) sensitivity and reduced the K(d) for free [Ca(2+)](i) from approximately 20 to 12 microM, but did not alter the slope or maximal P(o). BAPTA, a fast Ca(2+) buffer, or an elevation in extracellular Ca(2+) concentration did not alter IP(3)-induced BK(Ca) channel activation. Heparin, an IP(3)R inhibitor, and a monoclonal type 1 IP(3)R (IP(3)R1) antibody blocked IP(3)-induced BK(Ca) channel activation. Adenophostin A, an IP(3)R agonist, also activated BK(Ca) channels. IP(3) activated BK(Ca) channels in inside-out patches from wild-type (IP(3)R1(+/+)) mouse arterial smooth muscle cells, but had no effect on BK(Ca) channels of IP(3)R1-deficient (IP(3)R1(-/-)) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP(3)R1 is located in close spatial proximity to BK(Ca) alpha subunits. The IP(3)R1 monoclonal antibody coimmunoprecipitated IP(3)R1 and BK(Ca) channel alpha and beta1 subunits from cerebral arteries. In summary, data indicate that IP(3)R1 activation elevates BK(Ca) channel apparent Ca(2+) sensitivity through local molecular coupling in arterial smooth muscle cells.

Figure 1.

IP₃ activates BK_Ca channels in cerebral artery smooth muscle cells. (A) Original recordings obtained from the same cell illustrating activation of BK_Ca channel by Bt-IP₃ at +60 mV. (B) Mean data (n: control, 5; 10 µM Bt-IP₃, 5; 50 µM Bt-IP₃, 5). *, P < 0.05.

Figure 2.

IP₃ activates BK_Ca channels in excised inside-out membrane patches. (A) Original recordings from the same inside-out patch illustrating concentration-dependent BK_Ca channel activation by IP₃ at −40 mV. (B) Mean data fit with a Boltzmann function. IP₃ increased BK_Ca channel P_o with an apparent K_d of 4.1 ± 1.3 µM, a slope of 2.8 ± 1.9, and a maximal P_o of 0.38 ± 0.02. Experimental numbers are ([IP₃]): 0.01 µM, 4; 0.1 µM, 4; 1 µM, 4; 3 µM, 5; 10 µM, 5; 30 µM, 5. (C) Mean data illustrating that buffering Ca²⁺ with BAPTA or elevating pipette (extracellular) Ca²⁺ from 10 µM to 2 mM does not alter IP₃ (10 µM)-induced BK_Ca channel activation (n: 10 µM [Ca²⁺]_o/10 µM [Ca²⁺]_i (EGTA/EGTA); control, 4; IP₃, 4; 10 µM [Ca²⁺]_o/10 µM [Ca²⁺]_i (BAPTA/BAPTA); control, 4; IP₃, 4; 2 mM [Ca²⁺]_o/10 µM [Ca²⁺]_i (none/EGTA) control, 3; IP₃, 3). *, P < 0.05.

Figure 3.

IP₃ elevates BK_Ca channel apparent Ca²⁺ sensitivity. (A) Mean data illustrating IP₃-induced BK_Ca channel activation over a range of free Ca²⁺ concentrations in inside-out patches at −40 mV. Data are fit with a Boltzmann function constrained to ≥0. Experimental numbers are given after each [Ca²⁺] (µM): control: 1, 8; 3, 6; 10, 6; 30, 6; 100, 8; 300, 8; 10 µM IP₃: 1, 7; 3, 6; 10, 6; 30, 6; 100, 7; 300, 7. (B) Mean data illustrating IP₃-induced change in BK_Ca channel P_o at each [Ca²⁺]. Data are fit with a Gaussian function. In control, the K_d for Ca²⁺ was 20.4 ± 0.9 µM, a slope of 1.2 ± 0.2, and a maximum P_o of 0.82 ± 0.04. 10 µM IP₃ decreased the mean K_d for Ca²⁺ to 12.4 ± 0.8 µM (P < 0.05), but did not alter the slope (1.3 ± 0.3) or the maximum P_o (0.83 ± 0.03; P > 0.05 for each).

Figure 4.

IP₃R1 mediates IP₃-induced BK_Ca channel activation. (A) Mean data illustrating that heparin reverses IP₃-induced BK_Ca channel activation (n: control (10 µM Ca²⁺), 5; 30 µM IP₃, 5; 30 µM IP₃ plus 1 mg/ml heparin, 5). (B) Original recording from the same inside-out patch illustrating control BK_Ca channel activity (i), IP₃ (30 µM)-induced channel activation (ii), reversal by monoclonal IP₃R1 antibody (iii, 1:100; clone L24/18; NeuroMab), and no further effect of heparin applied in the continued presence of IP₃R1 antibody (iv). (C) Mean data illustrating that monoclonal IP₃R1 antibody inhibits IP₃-induced BK_Ca channel activation, and that the addition of heparin leads to no further change in activity (control (10 µM Ca²⁺), n = 4; 30 µM IP₃, n = 4; 30 µM IP₃ plus IP₃R1 antibody (1:100), n = 4; 30 µM IP₃ plus IP₃R1 antibody (1:100), n = 4; 30 µM IP₃ plus IP₃R1 antibody (1:100) plus heparin, n = 4; IP₃ plus boiled IP₃R1 antibody, n = 4). (D) Mean data illustrating that adenophostin A activates BK_Ca channels (n: control (10 µM Ca²⁺), 4; 1 µM adenophostin A, 4). *, P < 0.05.

Figure 5.

Genetic ablation of IP₃R1 abolishes IP₃-induced BK_Ca channel activation in cerebral artery smooth muscle cells. (A) Western blot indicating that IP₃R1 protein (∼270 kD) is present in IP₃R1^+/+ mouse aorta and absent in IP₃R1^−/− aorta. (B) Mean data illustrating that 10 µM IP₃ activates BK_Ca channels in inside-out patches from mouse IP₃R1^+/+ cerebral artery smooth muscle cells, but not in mouse IP₃R1^−/− cerebral artery smooth muscle cells. Mean data also indicate that 1 mg/ml heparin reverses IP₃-induced BK_Ca channel activation in IP₃R1^+/+ cells. Membrane voltage was −40 mV. IP₃R1^−/−: control, n = 5; IP₃, n = 5; IP₃ plus heparin, n = 5. IP₃R1^+/+: control, n = 5; IP₃, n = 5; IP₃ plus heparin, n = 5. *, P < 0.05.

Figure 6.

BK_Ca channel subunits are located in close spatial proximity to IP₃R1 in cerebral artery smooth muscle cells. (A) Immuno-FRET data illustrating close spatial proximity of IP₃R1 to BK_Ca channel α subunits. Cy2- and Cy3-labeled secondary antibodies bound to IP₃R1 and BK_Ca channels generate N-FRET. In contrast, secondary antibodies bound to IP₃R1 and TRPM4 do not generate significant N-FRET. Bars, 10 µm. (B) Monoclonal IP₃R1 antibody coimmunoprecipitates IP₃R1 (∼270 kD), BK_Ca channel α (∼125 kD), and β1 (∼36 kD) subunits from cerebral arteries. Lysate supernatant was used as the input (+ve) control. Beads that have no antibody-binding capacity were used in the negative (−ve) coIP control.