Bisphenol A activates Maxi-K (K(Ca)1.1) channels in coronary smooth muscle

Abstract

Background and purpose: Bisphenol A (BPA) is used to manufacture plastics, including containers for food into which it may leach. High levels of exposure to this oestrogenic endocrine disruptor are associated with diabetes and heart disease. Oestrogen and oestrogen receptor modulators increase the activity of large conductance Ca(2+)/voltage-sensitive K(+) (Maxi-K; K(Ca)1.1) channels, but the effects of BPA on Maxi-K channels are unknown. We tested the hypothesis that BPA activates Maxi-K channels through a mechanism that depends upon the regulatory beta1 subunit.

Experimental approach: Patch-clamp recordings of Maxi-K channels were made in human and canine coronary smooth muscle cells as well as in AD-293 cells expressing pore-forming alpha or alpha plus beta1 subunits.

Key results: BPA (10 microM) activated an outward current in smooth muscle cells that was inhibited by penitrem A (1 microM), a Maxi-K blocker. BPA increased Maxi-K activity in inside-out patches from coronary smooth muscle, but had no effect on single channel conductance. In AD-293 cells with Maxi-K channels composed of alpha subunits alone, 10 microM BPA did not affect channel activity. When channels in AD-293 cells contained beta1 subunits, 10 microM BPA increased channel activity. Effects of BPA were rapid (<1 min) and reversible. A higher concentration of BPA (100 microM) increased Maxi-K current independent of the beta1 subunit.

Conclusions and implications: Our data indicate that BPA increased the activity of Maxi-K channels and may represent a basis for some potential toxicological effects.

Figure 1

Bisphenol A (BPA) increases large conductance Ca²⁺/voltage-sensitive K⁺ channel (Maxi-K) current in smooth muscle cells cultured from the canine coronary artery. (A) Representative currents are shown under control conditions, with the application of 10 µM BPA, and with 1 µM penitrem A. (B) Currents from a representative cell are shown under control conditions, with 1 µM penitrem A, and with 10 µM BPA in the continued presence of penitrem A. (C) The voltage template used to elicit currents in this and subsequent figures. (D) Group data (n= 5) demonstrate the sensitivity of currents to BPA and penitrem A in cultured coronary artery smooth muscle cells. (E) Penitrem A prevented BPA-induced increase in Maxi-K current (n= 4). *P < 0.05 versus control by one-way anova.

Figure 2

Bisphenol A (BPA) increases large conductance Ca²⁺/voltage-sensitive K⁺ (Maxi-K) channel the NP_o (number of channels × open probability) in smooth muscle cells freshly isolated from the canine coronary artery. (A) Representative 1 min recordings of Maxi-K channel activity in an inside-out patch before and after exposure to 10 µM BPA are shown. Patch potential was +80 mV and currents were recorded in symmetrical 140 mM K⁺ with 100 nM free Ca²⁺. BPA increased NP_o, but had no effect on single channel amplitude. (B) Group data (n= 10 cells from 4 dogs) demonstrate that 10 µM BPA increases NP_o. *P < 0.05 by paired t-test. (C) Group data (n= 10) show that BPA has no effect on single conductance.

Figure 3

Large conductance Ca²⁺/voltage-sensitive K⁺ channel (Maxi-K) β1 subunit confers sensitivity to 10 µM bisphenol A (BPA). Currents were measured from AD-293 cells transfected with green fluorescent protein (GFP) (A) and Maxi-K channels composed of α (B) or α+β1 (C) subunits. Voltage template used is shown in Figure 1C. Very little whole-cell current was observed in cells expressing GFP only. Cells transfected with the α subunit demonstrated large outward currents with fast activation and little or no inactivation. Cells expressing α+β1 subunits demonstrated large outward currents with slower activation. (D) Records from a representative cell transfected with the Maxi-K α subunit show that current is unaffected by 10 µM BPA, but blocked by 1 µM penitrem A. (E) Current in a cell transfected with α+β1 subunits increased with application of 10 µM BPA and was blocked by 1 µM penitrem A. (F and G) Group data illustrate the effect of 10 µM BPA and 1 µM penitrem A on current in cells expressing α (n= 11) or α+β1 (n= 7) subunits. *P < 0.05 versus control by two-way repeated measures anova.

Figure 4

Bisphenol A (BPA) (10 µM) increases NP_o (number of channels × open probability) of large conductance Ca²⁺/voltage-sensitive K⁺ (Maxi-K) channels containing the β1 subunit. Representative 1 s recordings of Maxi-K channel activity in inside-out patches from AD-293 cells expressing Maxi-K α (A) or α+β1 (B) subunits before and after exposure to 10 µM BPA. Patch potential was +40 mV in symmetrical 140 mM K⁺ solutions containing 100 nM free Ca²⁺. (C and D) All-points amplitude histograms derived from 1 min recordings of patches shown in (A and B). BPA increased the NP_o of Maxi-K channels containing the β1 subunit and had no effect on single channel conductance.

Figure 5

The activation of large conductance Ca²⁺/voltage-sensitive K⁺ (Maxi-K) channels by bisphenol A (BPA) is concentration-dependent and reversible. (A) Maxi-K currents were measured in the whole-cell configuration after stepping membrane potential from −80 to +100 mV. Cells expressing Maxi-K channels composed of α or α+β1 subunits were studied (n= 7–12) under control conditions and after exposure to 1, 10 and 100 µM BPA. Current was increased relative to control at all three concentrations in cells expressing α+β1 subunits; current was increased only with 100 µM BPA in cells expressing the α subunit alone. *Significant differences between α and α+β1 by two-way anova. (B) Maxi-K (α) and Maxi-K (α+β1) currents were elicited by stepping the membrane potential from −80 to +100 mV for 300 ms every 10 s. BPA (10 µM) was applied for 2 min and washed out. Current increased rapidly with exposure to BPA and returned towards baseline with washout (n= 4).

Figure 6

Bisphenol A (BPA), at a concentration of 100 µM, increases large conductance Ca²⁺/voltage-sensitive K⁺ channel (Maxi-K) current in human coronary artery smooth muscle cells. (A) Representative records demonstrate that BPA (100 µM) increased whole-cell current in smooth muscle cells cultured from human coronary arteries. (B) Contains group data (n= 3–7) for effect of three concentrations of BPA on Maxi-K current. (C) RT-PCR revealed the expression of KCNMA1 (α subunit; 119 bp product) but not KCNMB1 (β1 subunit; 357 bp product) in cultured human smooth muscle cells. Human brain cDNA was used as a control, and the expression of KCNMA1 and KCNMB1 was detected. –RT is a reaction without reverse transcriptase; NTC is a ‘no template’ control.