Alex M. Dopico

Ultra‐potent synthetic opioids are powerful analgesics but also a leading cause of drug overdose deaths. Calcium−/voltage‐gated potassium channels of large conductance (BK) control vital physiological processes, with their accessory β1 (encoded by Kcnmb1 in mice) subunit being predominantly expressed in smooth muscle. Thus, we hypothesize that BK β1 determines the effect of fentanyl on cerebral artery diameter. Here we report that fentanyl (0.001–10 μ m ) activates BK heteromers consisting of slo1 + β1 subunits in CHO cells, but not homomers of slo1. In ex vivo pressurized mouse cerebral arteries, pharmacological inhibition of BK or β1 genetic deletion ( Kcnmb1 ^−/− ) led to fentanyl‐induced vasoconstriction. Naloxone, an opioid receptor antagonist, ablates the fentanyl response in wild‐type cerebral arteries whereas mild constriction persists in Kcnmb 1 ^−/− arteries. Therefore, we identify BK β1 as a functional fentanyl target which acts independently of opioid receptors to regulate fentanyl action on cerebral artery diameter.

Calcium- and voltage-gated potassium channels of large conductance (BK channels) in smooth muscle (SM) act as part of a negative feedback mechanism on SM contraction and associated decrease in cerebral artery diameter. Functional BK channels result from tetrameric association of α subunits encoded by KCNMA1 (Slo1). Ionic current from slo1 channels is inhibited by cholesterol in artificial lipid bilayers, an effect significantly reduced by the slo1 Y450F substitution. Whether such substitution affects cholesterol action on cerebral artery SM BK channel function and diameter remains unknown. Using the KCNMA1Y450F knock-in (K/I) mouse, we determined the effect of cholesterol enrichment on BK currents in native SM cells from middle cerebral artery using patch-clamp electrophysiology and the artery diameter ex vivo response to cholesterol. Results show that the KCNMA1Y450F K/I mutation modifies both basal function and the channel’s response to cholesterol enrichment. Such modifications are detectable solely in SM cells from males, demonstrating sexual dimorphism. Unexpectedly, the modifications introduced by the Y450F substitution do not translate into observable changes in middle cerebral artery diameter ex vivo, suggesting that mechanisms at the SM level compensate for changes driven by the KCNMA1 point mutation under study.

Potassium homeostasis is essential for a variety of physiological functions, many of which are critically dependent on proper function and expression of potassium channels. Though only a small portion of the large potassium channel family, voltage- and calcium-gated potassium channels of large conductance (MaxiK, BK) are ubiquitously expressed in the human body, and contribute to potassium homeostasis. Of particular interest, BK channels are major regulators of cerebral artery smooth muscle tone and diameter. BK-mediated currents are inhibited by many endogenous ligands including cholesterol, a major component of mammalian cell membranes and regulator of membrane protein function. Upon dietary intake of cholesterol-rich foods, cholesterol levels in cerebrovascular smooth muscle are increased. While cholesterol causes plaque formation in the lining of many artery types throughout the human body, cerebral arteries are rather resilient to this process, which is likely due to active uptake of cholesterol into the vascular smooth muscle layer. Upon cholesterol accumulation in cerebrovascular smooth muscle cells, BK channel regulation of myogenic tone is altered, and can exacerbate cholesterol-driven pathologies. In this chapter, we review BK channels, cholesterol interactions with these channels, and the influence of such interactions on cerebral artery function.

Toluene intoxication constitutes a persistent public health problem worldwide. While most organs can be damaged, the brain is a primary target whether exposure is accidental, occupational, or recreational. Interventions to prevent/revert brain damage by toluene are curtailed by the scarce information on the molecular targets and mechanisms mediating toluene's brain toxicity and the common exposure to other neurotoxins and/or coexistence of neurological/psychiatric disorders. We examine ( a ) the physicochemical properties of toluene that allow this inhalant to primarily target the lipid-rich brain; ( b ) the cell types targeted by toluene (neurons, different types of glia), while considering a cerebrovascular component; and ( c ) putative molecular mechanisms by which toluene may modify receptor function while analyzing structural features that allow toluene to directly interact with membrane lipids or specific proteins. This information constitutes a stepping stone to design pharmacotherapies that counteract toluene brain intoxication.

Moderate-to-heavy episodic (“binge”) drinking is the most common form of alcohol consumption in the United States. Alcohol at binge drinking concentrations reduces brain artery diameter in vivo and in vitro in many species including rats, mice, and humans. Despite the critical role played by brain vessels in maintaining neuronal function, there is a shortage of methodologies to simultaneously assess neuron and blood vessel function in deep brain regions. Here, we investigate cerebrovascular responses to ethanol by choosing a deep brain region that is implicated in alcohol disruption of brain function, the hippocampal CA1, and describe the process for obtaining simultaneous imaging of pyramidal neuron activity and diameter of nearby microvessels in freely moving mice via a dual-color miniscope. Recordings of neurovascular events were performed upon intraperitoneal injection of saline versus 3 g/kg ethanol in the same mouse. In male mice, ethanol mildly increased the amplitude of calcium signals while robustly decreasing their frequency. Simultaneously, ethanol decreased microvessel diameter. In females, ethanol did not change the amplitude or frequency of calcium signals from CA1 neurons but decreased microvessel diameter. A linear regression of ethanol-induced reduction in number of active neurons and microvessel constriction revealed a positive correlation ( R = 0.981) in females. Together, these data demonstrate the feasibility of simultaneously evaluating neuronal and vascular components of alcohol actions in a deep brain area in freely moving mice, as well as the sexual dimorphism of hippocampal neurovascular responses to alcohol.

Progesterone (≥1 µM) is used in recovery of cerebral ischemia, an effect likely contributed to by cerebrovascular dilation. The targets of this progesterone action are unknown. We report that micromolar (µM) progesterone activates mouse cerebrovascular myocyte BK channels; this action is lost in β₁^-/- mice myocytes and in lipid bilayers containing BK α subunit homomeric channels but sustained on β₁/β₄-containing heteromers. Progesterone binds to both regulatory subunits, involving two steroid binding sites conserved in β₁-β₄: high-affinity (sub-µM), which involves Trp87 in β₁ loop, and low-affinity (µM) defined by TM1 Tyr32 and TM2 Trp163. Thus progesterone, but not its oxime, bridges TM1-TM2. Mutation of the high-affinity site blunts channel activation by progesterone underscoring a permissive role of the high-affinity site: progesterone binding to this site enables steroid binding at the low-affinity site, which activates the channel. In support of our model, cerebrovascular dilation evoked by μM progesterone is lost by mutating Tyr32 or Trp163 in β₁ whereas these mutations do not affect alcohol-induced cerebrovascular constriction. Furthermore, this alcohol action is effectively counteracted both in vitro and in vivo by progesterone but not by its oxime.

Despite the significant number of people who may be taking pregnenolone supplements while drinking alcohol (ethanol), the widely documented cerebrovascular actions of pregnenolone and ethanol, and the critical dependence of cerebrovascular function on cerebral artery diameter, there are no studies addressing the effect of pregnenolone + ethanol in combination on cerebral artery diameter. We investigated this by evaluating the effect of this combination on middle cerebral artery diameter in male and female C57BL/6J mice, both in vivo and in vitro. The use of de-endothelialized, in vitro pressurized middle cerebral artery segments allowed us to conduct a concentration-response study of constriction induced by pregnenolone ± ethanol, in which drug action could be evaluated independently of circulating and endothelial factors. In both male and female animals, pregnenolone at lower concentrations (≤1 µM) was found to synergize with 50 mM ethanol to cause vasoconstriction. In both sexes, this synergism was lost as one or both vasoconstrictors approached their maximally effective concentrations (75 mM and 10 µM for ethanol and pregnenolone, respectively), whether this was evaluated in vitro or in vivo using a cranial window. Vasoconstriction by pregnenolone + ethanol was abolished by 1 µM paxilline, indicating BK channel involvement. Moreover, cell-free recordings of BK channel activity in cerebral artery myocyte membranes showed that 10 µM pregnenolone and pregnenolone +50 mM ethanol reduced channel activity to an identical extent, suggesting that these drugs inhibit cerebrovascular BK channels via a common mechanism or mechanisms. Indeed, pregnenolone was found to disrupt allosteric coupling to Ca²⁺-driven gating, as previously reported for ethanol.

Calcium/voltage-activated potassium channels (BK) control smooth muscle (SM) tone and cerebral artery diameter. They include channel-forming α and regulatory β₁ subunits, the latter being highly expressed in SM. Both subunits participate in steroid-induced modification of BK activity: β₁ provides recognition for estradiol and cholanes, resulting in BK potentiation, whereas α suffices for BK inhibition by cholesterol or pregnenolone. Aldosterone can modify cerebral artery function independently of its effects outside the brain, yet BK involvement in aldosterone’s cerebrovascular action and identification of channel subunits, possibly involved in steroid action, remains uninvestigated. Using microscale thermophoresis, we demonstrated that each subunit type presents two recognition sites for aldosterone: at 0.3 and ≥10 µM for α and at 0.3–1 µM and ≥100 µM for β₁. Next, we probed aldosterone on SM BK activity and diameter of middle cerebral artery (MCA) isolated from β₁^−/− vs. wt mice. Data showed that β₁ leftward-shifted aldosterone-induced BK activation, rendering EC₅₀~3 μM and EC_MAX ≥ 10 μM, at which BK activity increased by 20%. At similar concentrations, aldosterone mildly yet significantly dilated MCA independently of circulating and endothelial factors. Lastly, aldosterone-induced MCA dilation was lost in β₁^−/− mice. Therefore, β₁ enables BK activation and MCA dilation by low µM aldosterone.