Beta1-subunit of the Ca2+-activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle

Abstract

1. The large-conductance calcium-activated potassium (BK) channel plays an important role in controlling membrane potential and contractility of urinary bladder smooth muscle (UBSM). These channels are composed of a pore-forming alpha-subunit and an accessory, smooth muscle-specific, beta1-subunit. 2. Our aim was to determine the functional role of the beta1-subunit of the BK channel in controlling the contractions of UBSM by using BK channel beta1-subunit 'knock-out' (KO) mice. 3. The beta-galactosidase reporter (lacZ gene) was targeted to the beta1 locus, which provided the opportunity to examine the expression of the beta1-subunit in UBSM. Based on this approach, the beta1-subunit is highly expressed in UBSM. 4. BK channels lacking beta1-subunits have reduced activity, consistent with a shift in BK channel voltage/Ca2+ sensitivity. 5. Iberiotoxin, an inhibitor of BK channels, increased the amplitude and decreased the frequency of phasic contractions of UBSM strips from control mice. 6. The effects of the beta1-subunit deletion on contractions were similar to the effect of iberiotoxin on control mice. The UBSM strips from beta1-subunit KO mice had elevated phasic contraction amplitude and decreased frequency when compared to control UBSM strips. 7. Iberiotoxin increased the amplitude and frequency of phasic contractions, and UBSM tone of UBSM strips from beta1-subunit KO mice, suggesting that BK channels still regulate contractions in the absence of the beta1-subunit. 8. The results indicate that the beta1-subunit, by modulating BK channel activity, plays a significant role in the regulation of phasic contractions of the urinary bladder.

Figure 1. Detection of lacZ gene expression from β1 gene-targeted mice

Whole urinary bladders isolated from β1-subunit KO and control mice were stained with X-gal staining solution to identify lacZ gene expression (blue colour). The smooth muscle layer of the bladder from the β1-subunit KO mouse shows intense and uniform lacZ expression.

Figure 2. Voltage dependence of BK channel activity in UBSM myocytes from control and β1-subunit KO mice

A, single-channel recordings from inside-out patches held at -40 and +40 mV in 10 μm free Ca²⁺. Arrows indicate the closed state of the channels. B, BK channel open probability in control and β1-subunit KO animals at two different voltages (-40 and +40 mV). Values are means ±s.e.m. Data were obtained from 9-11 cells of each animal group. *P < 0.05, **P < 0.005.

Figure 3. Single BK channel conductance and channel density in UBSM cells from β1-subunit KO and control mice

A, current-voltage relationship for the single BK channel conductance. Mean amplitude ±s.e.m. of the BK channel unitary current from β1-subunit KO and control mice is plotted versus the membrane potential. The standard error bars are present but are too small to be seen in the figure. B, mean number of channels per patch excised from UBSM cells from control and β1-subunit KO mice (n = 11).

Figure 5. Differences in phasic contractions of UBSM strips from β1-subunit KO and control mice

A, comparison between 20 mm K⁺-induced phasic contractions of a UBSM strip from a control mouse (in the absence and presence of iberiotoxin) and from a β1-subunit KO mouse. B, differences in the phasic contraction frequency of UBSM strips from control mice, control mice treated with iberiotoxin (100 nm), and β1-subunit KO mice in response to 20 mm K⁺ and 1 μm carbachol. C, differences in the phasic contraction amplitude of UBSM strips from control mice, control mice treated with iberiotoxin (100 nm), and β1-subunit KO mice in response to 20 mm K⁺ and 1 μm carbachol. Values are means ±s.e.m. In C, data were normalized to the 60 mm K⁺-induced response. *P < 0.05, **P < 0.01.

Figure 4. Original recordings of spontaneous, 20 mm K⁺-induced and 1 μm carbachol-induced phasic contractions of UBSM strips isolated from control mice and the effect of blocking BK channels with iberiotoxin

Iberiotoxin (100 nm) increased the amplitude but decreased the frequency of the phasic contractions.

Figure 6. Original recordings of phasic contractions from UBSM strips isolated from β1-subunit KO mice and the effect of blocking BK channels with iberiotoxin

Iberiotoxin (100 nm) increased the amplitude and frequency of the spontaneous 20 mm K⁺-induced and 1 μm carbachol-induced phasic contractions of UBSM strips isolated from β1-subunit KO mice.