Ibra Fancher

Diabetes mellitus reduces the function and expression of ATP-dependent K⁺ channels in cardiac mitochondria.

AIM Our goal was to determine the effects of type I diabetes mellitus on the function and expression of ATP-dependent K(+) channels in cardiac mitochondria (mitoKATP), composed of a pore-forming subunit (Kir6.1) and a diazoxide-sensitive sulphonylurea receptor (SUR1). We tested the hypothesis that diabetes reduces Kir6.1 and SUR1 expression as well as diazoxide-induced depolarization of mitochondrial membrane potential (ΔΨm). MAIN METHODS Male FVB mice were made diabetic for 5weeks with multiple low dose injections of streptozotocin. Cardiac mitochondria were separated into two populations: subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). mitoKATP expression was determined via Western blot analysis of Kir6.1 and SUR1 proteins. mitoKATP function was determined by measuring ΔΨm with the potentiometric dye rhodamine 123. KEY FINDINGS Diabetes reduced Kir6.1 and SUR1 expression in IFM by over 40% (p<0.05 for both). Similarly, diabetes reduced Kir6.1 expression in SSM by approximately 40% (p<0.05); however, SUR1 expression was unaffected. Opening mitoKATP with diazoxide (100μM) depolarized control IFM ΔΨm by 80% of the valinomycin maximum; diabetic IFM depolarized only 30% (p<0.05). Diazoxide-induced depolarization was much less in SSM (20-30%) and unaffected by diabetes. SIGNIFICANCE Our data indicate that diabetes reduces mitoKATP expression and function in IFM. These changes in mitoKATP may provide an opportunity to understand mechanisms leading to diabetic cardiomyopathy and loss of cardioprotective mechanisms in the diabetic heart.

Penitrem A as a Tool for Understanding the Role of Large Conductance Ca2+/Voltage-Sensitive K+ Channels in Vascular Function

Large conductance, Ca2+/voltage-sensitive K+ channels (BK channels) are well characterized, but their physiological roles, often determined through pharmacological manipulation, are less clear. Iberiotoxin is considered the “gold standard” antagonist, but cost and membrane-impermeability limit its usefulness. Economical and membrane-permeable alternatives could facilitate the study of BK channels. Thus, we characterized the effect of penitrem A, a tremorigenic mycotoxin, on BK channels and demonstrate its utility for studying vascular function in vitro and in vivo. Whole-cell currents from human embryonic kidney 293 cells transfected with hSlo α or α + β1 were blocked >95% by penitrem A (IC50 6.4 versus 64.4 nM; p < 0.05). Furthermore, penitrem A inhibited BK channels in inside-out and cell-attached patches, whereas iberiotoxin could not. Inhibitory effects of penitrem A on whole-cell K+ currents were equivalent to iberiotoxin in canine coronary smooth muscle cells. As for specificity, penitrem A had no effect on native delayed rectifier K+ currents, cloned voltage-dependent Kv1.5 channels, or native ATP-dependent KATP current. Penitrem A enhanced the sensitivity to K+-induced contraction in canine coronary arteries by 23 ± 5% (p < 0.05) and increased the blood pressure response to phenylephrine in anesthetized mice by 36 ± 11% (p < 0.05). Our data indicate that penitrem A is a useful tool for studying the role of BK channels in vascular function and is practical for cell and tissue (in vitro) studies as well as anesthetized animal (in vivo) experiments.

Bisphenol A activates BK channels through effects on α and β1 subunits.

We demonstrated previously that BK (KCa1.1) channel activity (NPo) increases in response to bisphenol A (BPA). Moreover, BK channels containing regulatory β1 subunits were more sensitive to the stimulatory effect of BPA. How BPA increases BK channel NPo remains mostly unknown. Estradiol activates BK channels by binding to an extracellular site, but neither the existence nor location of a BPA binding site has been demonstrated. We tested the hypothesis that an extracellular binding site is responsible for activation of BK channels by BPA. We synthesized membrane-impermeant BPA-monosulfate (BPA-MS) and used patch clamp electrophysiology to study channels composed of α or α + β1 subunits in cell-attached (C-A), whole-cell (W-C), and inside-out (I-O) patches. In C-A patches, bath application of BPA-MS (100 μM) had no effect on the NPo of BK channels, regardless of their subunit composition. Importantly, however, subsequent addition of membrane-permeant BPA (100 μM) increased the NPo of both α and α + β1 channels in C-A patches. The C-A data indicate that in order to alter BK channel NPo, BPA must interact with the channel itself (or some closely associated partner) and diffusible messengers are not involved. In W-C patches, 100 μM BPA-MS activated current in cells expressing α subunits, whereas cells expressing α + β1 subunits responded similarly to a log-order lower concentration (10 μM). The W-C data suggest that an extracellular activation site exists, but do not eliminate the possibility that an intracellular site may also be present. In I-O patches, where the cytoplasmic face was exposed to the bath, BPA-MS had no effect on the NPo of BK α subunits, but BPA increased it. BPA-MS increased the NPo of α + β1 channels in I-O patches, but not as much as BPA. We conclude that BPA activates BK α via an extracellular site and that BPA-sensitivity is increased by the β1 subunit, which may also constitute part of an intracellular binding site.

Diphenyl Phosphine Oxide‐1‐Sensitive K+ Channels Contribute to the Vascular Tone and Reactivity of Resistance Arteries From Brain and Skeletal Muscle

Many types of vascular smooth muscle cells exhibit prominent KDR currents. These KDR currents may be mediated, at least in part, by KV1.5 channels, which are sensitive to inhibition by DPO‐1. We tested the hypothesis that DPO‐1‐sensitive KDR channels regulate the tone and reactivity of resistance‐sized vessels from rat brain (MCA) and skeletal muscle (GA).