Coronary arterial BK channel dysfunction exacerbates ischemia/reperfusion-induced myocardial injury in diabetic mice.

The large conductance Ca(2+)-activated K(+) (BK) channels, abundantly expressed in coronary artery smooth muscle cells (SMCs), play a pivotal role in regulating coronary circulation. A large body of evidence indicates that coronary arterial BK channel function is diminished in both type 1 and type 2 diabetes. However, the consequence of coronary BK channel dysfunction in diabetes is not clear. We hypothesized that impaired coronary BK channel function exacerbates myocardial ischemia/reperfusion (I/R) injury in streptozotocin-induced diabetic mice. Combining patch-clamp techniques and cellular biological approaches, we found that diabetes facilitated the colocalization of angiotensin II (Ang II) type 1 receptors and BK channel α-subunits (BK-α), but not BK channel ß1-subunits (BK-ß1), in the caveolae of coronary SMCs. This caveolar compartmentation in vascular SMCs not only enhanced Ang II-mediated inhibition of BK-α but also produced a physical disassociation between BK-α and BK-ß1, leading to increased infarct size in diabetic hearts. Most importantly, genetic ablation of caveolae integrity or pharmacological activation of coronary BK channels protected the cardiac function of diabetic mice from experimental I/R injury in both in vivo and ex vivo preparations. Our results demonstrate a vascular ionic mechanism underlying the poor outcome of myocardial injury in diabetes. Hence, activation of coronary BK channels may serve as a therapeutic target for cardiovascular complications of diabetes.

Effects of the novel BK (KCa 1.1) channel opener GoSlo-SR-5-130 are dependent on the presence of BKß subunits.

BACKGROUND AND PURPOSE: GoSlo-SR compounds are efficacious BK (KCa 1.1) channel openers, but little is known about their mechanism of action or effect on bladder contractility. We examined the effects of two closely related compounds on BK currents and bladder contractions. EXPERIMENTAL APPROACH: A combination of electrophysiology, molecular biology and synthetic chemistry was used to examine the effects of two novel channel agonists on BK channels from bladder smooth muscle cells and in HEK cells expressing BKα alone or in combination with either ß1 or ß4 subunits. KEY RESULTS: GoSlo-SR-5-6 shifted the voltage required for half maximal activation (V1/2 ) of BK channels approximately -100 mV, irrespective of the presence of regulatory ß subunits. The deaminated derivative, GoSlo-SR-5-130, also shifted the activation V1/2 in smooth muscle cells by approximately -100 mV; however, this was reduced by ∼80% in HEK cells expressing only BKα subunits. When ß1 or ß4 subunits were co-expressed with BKα, efficacy was restored. GoSlo-SR-5-130 caused a concentration-dependent reduction in spontaneous bladder contraction amplitude and this was abolished by iberiotoxin, consistent with an effect on BK channels. CONCLUSIONS AND IMPLICATIONS: GoSlo-SR-5-130 required ß1 or ß4 subunits to mediate its full effects, whereas GoSlo-SR-5-6 worked equally well in the absence or presence of ß subunits. GoSlo-SR-5-130 inhibited spontaneous bladder contractions by activating BK channels. The novel BK channel opener, GoSlo-SR-5-130, is approximately fivefold more efficacious on BK channels with regulatory ß subunits and may be a useful scaffold in the development of drugs to treat diseases such as overactive bladder.

Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein.

Large-conductance voltage- and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstruction-induced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC(20)) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel alpha- and beta-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BKbeta mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK beta-subunit was greater than that of the BK alpha-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK beta-subunit was employed to study the effect of BK depletion on MLC(20) phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC(20) phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC(20) phosphorylation.

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

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.

Channel beta2-4 subunits fail to substitute for beta1 in sensitizing BK channels to lithocholate.

Large conductance, calcium- and voltage-gated potassium (BK) channels regulate numerous physiological processes. While most basic functional characteristics of native BK channels are reproduced by BK alpha (slo1) subunit homotetramers, key biophysical and pharmacological properties are drastically modified by the presence of auxiliary beta subunits (encoded by KCNMB1-4). Numerous physiological steroids, including sex hormones, gluco- and mineralocorticoids, activate beta subunit-containing BK channels, yet these steroids appear to be sensed by different types of beta subunits, with some steroids being sensed by homomeric slo1 channels as well. We recently showed that beta1 sensitizes the BK channel to microM concentrations of lithocholate (LC). Following expression of rat cerebral artery myocyte slo1 subunits ("cbv1") with beta1, beta2, beta3 or beta4 in Xenopus laevis oocytes we now demonstrate that BK beta2, beta3 and beta4 subunits fail to substitute for beta1 in providing LC-sensitivity (150 microM) to the BK channel. These findings document for the first time a rather selective steroid activation of BK channels via a particular channel accessory subunit. In addition, LC routinely activated native BK channels in myocytes freshly isolated from rat cerebral artery smooth muscle, where BK beta1 is highly expressed, while failing to do so in skeletal (flexor digitorum brevis) muscle, where BK beta1 expression is negligible. This indicates that the native environment of the BK channel sustains the LC-sensitivity distinctly provided to the BK channel by beta1 subunits. Our study indicates that LC represents a unique tool to probe the presence of functional beta1-subunits and selectively activate BK channels in tissues that highly express KCNMB1.

Acetic acid opens large-conductance Ca2+-activated K+ channels in guinea pig detrusor smooth muscle cells.

Acetic acid was found to have actions on urinary bladder smooth muscle in our routine ion channel screening assays. Numerous studies have examined the mechanisms of bladder irritation by acetic acid; however, the direct effect of acetic acid on ion channels in detrusor smooth muscle cells has not been evaluated. We used whole-cell patch-clamp techniques to examine the effect of acetic acid on large-conductance Ca2+-activated K+ channels (BKCa) from guinea pig detrusor smooth muscle cells and CHO cells expressing recombinant human BKCaalphabeta1 (CHO BKCaalphabeta1) and human BKCaalpha (CHO BKCaalpha). Acetic acid activated BKCa currents in a concentration-dependent (0.01% to 0.05% v/v) manner in all the cell systems studied. Acetic acid (0.05%) increased BKCa current at +30 mV by 2764+/-918% (n=8) in guinea pig detrusor smooth muscle cells. Acetic acid (0.03%) shifted the V1/2 of conductance-voltage curve by 64+/-14 (n=5), 128+/-14 (n=5), and 126+/-12 mV (n=4) in CHO BKCaalpha, CHO BKCaalphabeta1 and detrusor smooth muscle cells, respectively. This effect of acetic acid was found to be independent of pH and was also not produced by its salt form, sodium acetate. Automated patch-clamp experiments also showed similar activation of CHO BKCaalphabeta1 by acetic acid. In conclusion, acetic acid directly activates BKCa channels in detrusor smooth muscle cells. This novel study necessitates caution while interpreting the results from acetic acid bladder irritation model.

Voltage-sensitive oxonol dyes are novel large-conductance Ca2+-activated K+ channel activators selective for beta1 and beta4 but not for beta2 subunits.

The large-conductance Ca(2+)-activated K(+) (BK) channel is activated by both the increase of intracellular Ca(2+) concentration and membrane depolarization. The BK channel plays crucial roles as a key molecule in the negative feedback mechanism regulating membrane excitability and cellular Ca(2+) in various cell types. Here, we report that a widely used slow-response voltage-sensitive fluorescent dye, bis(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(4)(3)], is a potent BK channel activator. The application of DiBAC(4)(3) at concentrations of 10 nM and higher significantly increased whole-cell BK channel currents in human embryonic kidney 293 cells expressing rat BK channel alpha and beta1 subunits (rBKalphabeta1). In the presence of 300 nM DiBAC(4)(3), the activation voltage of the BK channel current shifted to the negative direction by approximately 30 mV, but the single-channel conductance was not affected. DiBAC(4)(3) activated whole-cell rBKalphabeta1 and rBKalphabeta4 currents in the same concentration range but partially blocked rBKalphabeta2 currents. The BK channel alpha subunit alone and some other types of K(+) channels examined were not markedly affected by 1 microM DiBAC(4)(3). Structure-activity relationship analyses revealed that a set of oxo- and oxoanion-moieties in two 1,3-dialkylbarbituric acids, which are conjugated by oligomethine, is the novel skeleton for the beta-subunit-selective BK channel-opening property of DiBAC(4)(3) and related oxonol compounds. This conjugated structure may be located stereochemically in one plane. These findings provide a molecular and structural basis for understanding the regulatory mechanism of BK channel activity by an auxiliary beta subunit and will be fundamental to the development of beta-selective BK channel openers.