Interleukin-4 activates large-conductance, calcium-activated potassium (BKCa) channels in human airway smooth muscle cells

Abstract

Large-conductance, calcium-activated potassium (BK(Ca)) channels are regulated by voltage and near-membrane calcium concentrations and are determinants of membrane potential and excitability in airway smooth muscle cells. Since the T helper-2 (Th2) cytokine, interleukin (IL)-4, is an important mediator of airway inflammation, we investigated whether IL-4 rapidly regulated BK(Ca) activity in normal airway smooth muscle cells. On-cell voltage clamp recordings were made on subconfluent, cultured human bronchial smooth muscle cells (HBSMC). Interleukin-4 (50 ng ml(-1)), IL-13 (50 ng ml(-1)) or histamine (10 microm) was added to the bath during the recordings. Immunofluorescence studies with selective antibodies against the alpha and beta1 subunits of BK(Ca) were also performed. Both approaches demonstrated that HBSMC membranes contained large-conductance channels (>200 pS) with both calcium and voltage sensitivity, all of which is characteristic of the BK(Ca) channel. Histamine caused a rapid increase in channel activity, as expected. A new finding was that perfusion with IL-4 stimulated rapid, large increases in BK(Ca) channel activity (77.2 +/- 63.3-fold increase, P < 0.05, n = 18). This large potentiation depended on the presence of external calcium. In contrast, IL-13 (50 ng ml(-1)) had little effect on BK(Ca) channel activity, but inhibited the effect of IL-4. Thus, HBSMC contain functional BK(Ca) channels whose activity is rapidly potentiated by the cytokine, IL-4, but not by IL-13. These findings are consistent with a model in which IL-4 rapidly increases near-membrane calcium concentrations to regulate BK(Ca) activity.

Figure 1. Characterization of BK_Ca channels

A, typical ‘on-cell’ recording from a single patch on a HBSMC, illustrating the voltage sensitivity in 5 μm Ca²⁺. In this and subsequent figures, C represents the closed state and O1, O2, O3 and O4 indicate the number of channels open. B, following these recordings, the patch was pulled from the cell to obtain an ‘inside-out’ recording, and the calcium sensitivity (held at +30 mV) was determined as shown. C, the slope conductance (γ) is computed from data from the recordings shown in A.

Figure 2. Expression of BK_Ca channels

The HBSMC grown on coverslips were fixed and stained with antibodies and fluorophores as described in the Methods. Red fluorophor represents staining against the BK_Ca channel α subunit. Green fluorophor represents staining against the BK_Ca channel β1 subunit. Blue fluorophor represents staining with DAPI to visualize cell nuclei. The scale bar represents a length of 5 microns.

Figure 3. BK_Ca channel responses to histamine

Typical on-cell recordings illustrating a portion of the 20 s control voltage step (top, left) and the same duration step after the bath application of 10 μm histamine (top, right). The bottom panels are the all-point histograms for the 20 s recordings shown above them with an indication of the open probability (P_o) derived from the histograms.

Figure 4. BK_Ca channel responses to IL-4

Typical on-cell recordings illustrating a portion of the 20 s control voltage step (A, top trace) and the same step after ~1 min after the bath application of 50 ng ml⁻¹ of IL-4 (A, bottom trace). The panels in B are the all-point histograms for the recordings shown above them with an indication of the average open probability (nP_o) derived from the histograms.

Figure 5. On-cell recordings illustrating the typical response to IL-4 stimulation

In this patch, there are 7 or more channels responding. In the top right panel, the figure has a cropped appearance only because the signal was unexpectedly too large for the scale set by the gain. Two adjacent voltage steps (gap ~1 s) have been joined to show the onset and offset of the response. In the bottom two panels, the slow recovery back to control levels for sequential voltage steps in the continued presence of IL-4 is shown.

Figure 6. Lack of BK_Ca channel responses to IL-13

Typical on-cell recording illustrating the lack of response to the application of IL-13 (50 ng ml⁻¹). The sequential voltage steps shown are less than 1 s apart (read from left to right and top to bottom).

Figure 7. BK_Ca channel responses to IL-4 in the presence of IL-13

A, on-cell recordings illustrating the response to IL-4 in the presence of IL-13. The sequential voltage steps shown are less than 1 s apart (read from top to bottom). B, BK_Ca channel activity is expressed as a percentage of control as cells are exposed to IL-13 (50 ng ml⁻¹) and then IL-4 (50 ng ml⁻¹) in the continued presence of IL-13 (mean ± s.e.m., n = 7 cells). C, from the same experiments, BK_Ca channel activity is expressed as a percentage of control for 2 cells exposed to IL-4 alone (see Figs 4 and 5).

Figure 8. Effect of serum starvation on BK_Ca channel responses to IL-4

Diary plots of activity in recordings from 4 starved cells (A) and 4 fed cells (B) in response to IL-4 (50 ng ml⁻¹). Each point is the average open probability (nP_o) for successive 2 s intervals of the recording. These are raw scores. No attempt has been made to normalize or synchronize the observed response to bath application of IL-4. The responses plotted were selected for the duration of the recordings.

Figure 9. BK_Ca channel requirement for extracellular calcium

A, on-cell recordings showing no response to IL-4 in a cell bathed in an extracellular solution (KRH solution) in which calcium has been replaced by an equivalent amount of barium. B, all-point histograms for the recordings shown in A, with an indication of the average open probability (nP_o) derived from the histograms. C, on-cell recordings from a different cell, illustrating the lack of response to IL-4 in a cell bathed in an extracellular solution (KRH solution) without calcium.