Molecular Mechanisms Underlying AlcoholInduced Cerebral Artery Smooth Muscle BK Channel Inhibition and Eventual Cerebral Vasoconstriction

Introduction and Rationale: Ethanol (EtOH) at concentrations obtained in circulation during moderate to heavy episodic drinking, such as during binge drinking (30-60 mM) causes cerebral vasoconstriction in many species, including humans. Using rodents as a model to study ethanolinduced cerebral artery constriction, our laboratory demonstrated that ethanol-induced cerebral artery constriction is due to drug-induced reduction of STOCs (Spontaneous Transient Outward Currents) in cerebral artery smooth muscle. In this tissue, STOCs result from the activity of large conductance, calcium-and voltage-gated potassium (BK) channels. Indeed, ethanol (50 mM) decreases the steady-state activity (NPo) of vascular myocyte BK channels leading to an increase in cerebral artery tone. In native tissues, functional BK channels are oligomers of four channel-forming slo1 subunits that are associated with small, accessory subunits (β1-4). β subunits do not form channels themselves but modify BK current phenotype, including its pharmacology. In particular, the vascular smooth muscle-abundant BK β1 subunit is required for ethanol to inhibit cerebral artery myocyte BK channels under physiological conditions of voltage and calcium. In contrast, the neuronally-predominant β4 subunit does not support this ethanol action. The molecular bases of ethanol-mediated inhibition of β1 subunit-containing BK channels and resulting cerebral vasoconstriction remain unknown. Objective: Identify the BK β1 subunit regions and β1 subunit-dependent channel gating mechanisms underlying ethanol-induced inhibition of cerebral artery smooth muscle BK channel inhibition and eventual cerebral artery constriction. Methods: Combination of recombinant DNA and other molecular biology in vitro approaches, patch-clamp electrophysiology, allosteric gating modeling, reversible permeabilization of arteries with cDNAs, and artery pressurization techniques. Results: Ethanol sensitivity of slo1 current is dependent on the channel’s activating ion, i.e., Ca2+ i. Moreover, ethanol-induced modification of activity of slo1 (cbv1) and heteromeric cbv1+β1 BK channels is primarily due to modulation of calcium-driven gating: in particular, increase in Ca2+ i affinity; decrease in allosteric interaction between 1) RCK and voltage sensing domains and 2) RCKs and pore gate domains. Ethanol facilitation of channel inhibition is favored by β1-and β2 but not β3 or β4 subunits. Consistent with the involvement of calciumdependent mechanism, the former two drastically increase the channel’s apparent calcium sensitivity whereas the latter fail to do so. Transmembrane domains of BK-β1 subunit are essential for ethanol-mediated inhibition of β1-containing BK channels. In particular, the second transmembrane domain of β1 subunit is necessary for both inhibition of β1-containing BK channels and cerebral artery constriction evoked by ethanol. Conclusion: BK β1 subunit TM2 enables ethanol-induced inhibition of β1-containing BK channels and cerebral artery constriction, with drug action on channel activity being dependent on modification of calcium-gating parameters

Activation of human smooth muscle BK channels by hydrochlorothiazide requires cell integrity and the presence of BK β1 subunit

Thiazide-like diuretics are the most commonly used drugs to treat arterial hypertension, with their efficacy being linked to their chronic vasodilatory effect. Previous studies suggest that activation of the large conductance voltage- and Ca2+-dependent K+ (BK) channel (Slo 1, MaxiK channel) is responsible for the thiazide-induced vasodilatory effect. But the direct electrophysiological evidence supporting this claim is lacking. BK channels can be associated with one small accessory β-subunit (β1–β4) that confers specific biophysical and pharmacological characteristics to the current phenotype. The β1-subunit is primarily expressed in smooth muscle cells (SMCs). In this study we investigated the effect of hydrochlorothiazide (HCTZ) on BK channel activity in native SMCs from human umbilical artery (HUASMCs) and HEK293T cells expressing the BK channel (with and without the β1-subunit). Bath application of HCTZ (10 µmol/L) significantly augmented the BK current in HUASMCs when recorded using the whole-cell configurations, but it did not affect the unitary conductance and open probability of the BK channel in HUASMCs evaluated in the inside-out configuration, suggesting an indirect mechanism requiring cell integrity. In HEK293T cells expressing BK channels, HCTZ-augmented BK channel activity was only observed when the β1-subunit was co-expressed, being concentration-dependent with an EC50 of 28.4 µmol/L, whereas membrane potential did not influence the concentration relationship. Moreover, HCTZ did not affect the BK channel current in HEK293T cells evaluated in the inside-out configuration, but significantly increases the open probability in the cell-attached configuration. Our data demonstrate that a β1-subunit-dependent mechanism that requires SMC integrity leads to HCTZ-induced BK channel activation.Instituto de Estudios Inmunológicos y Fisiopatológico

Both Transmembrane Domains of BK β1 Subunits Are Essential to Confer the Normal Phenotype of β1-Containing BK Channels

<div><p>Voltage/Ca²⁺_i-gated, large conductance K⁺ (BK) channels result from tetrameric association of α (slo1) subunits. In most tissues, BK protein complexes include regulatory β subunits that contain two transmembrane domains (TM1, TM2), an extracellular loop, and two short intracellular termini. Four BK β types have been identified, each presenting a rather selective tissue-specific expression profile. Thus, BK β modifies current phenotype to suit physiology in a tissue-specific manner. The smooth muscle-abundant BK β1 drastically increases the channel's apparent Ca²⁺_i sensitivity. The resulting phenotype is critical for BK channel activity to increase in response to Ca²⁺ levels reached near the channel during depolarization-induced Ca²⁺ influx and myocyte contraction. The eventual BK channel activation generates outward K⁺ currents that drive the membrane potential in the negative direction and eventually counteract depolarization-induced Ca²⁺ influx. The BK β1 regions responsible for the characteristic phenotype of β1-containing BK channels remain to be identified. We used patch-clamp electrophysiology on channels resulting from the combination of smooth muscle slo1 (cbv1) subunits with smooth muscle-abundant β1, neuron-abundant β4, or chimeras constructed by swapping β1 and β4 regions, and determined the contribution of specific β1 regions to the BK phenotype. At Ca²⁺ levels found near the channel during myocyte contraction (10 µM), channel complexes that included chimeras having both TMs from β1 and the remaining regions (“background”) from β4 showed a phenotype (V_half, τ_act, τ_deact) identical to that of complexes containing <i>wt</i> β1. This phenotype could not be evoked by complexes that included chimeras combining either β1 TM1 or β1 TM2 with a β4 background. Likewise, β “halves” (each including β1 TM1 or β1 TM2) resulting from interrupting the continuity of the EC loop failed to render the normal phenotype, indicating that physical connection between β1 TMs <i>via</i> the EC loop is also necessary for proper channel function.</p></div

Schematic structure of β1 subunit-containing BK channel.

<p>Cartoon showing a slo1-β1 subunit heterodimer. The channel-forming slo1 subunit includes transmembrane (TM) segments S0-S6 and intracellular Regulatory of Conductance for K⁺ (RCK) domains, these domains including distinct residues that participate in sensing changes in Ca²⁺_i. Four slo1 monomers assemble to render fully functional BK channels. All four types of β subunits identified so far contain a similar design that includes intracellular N- and C-terminals, two transmembrane domains (TM1 and TM2), and an EC loop. <i>EC</i> and <i>IC</i> correspond to the extracellular and intracellular sides of the membrane.</p

Physical continuity of the EC loop between TM1 and TM2 is essential to confer the characteristic phenotype of β1-containing BK channel complexes.

<p>(A) Cartoons depicting two hβ1/hβ4 chimeric constructs termed “N-half” and “C-half”, obtained by cleaving the EC loop between TM1 and TM2 in the β4TMs₁ chimera. Regions of β1 and β4 are given in black and grey, respectively. When expressed together (panels C-G and main text), “N-half” and “C-half” chimera have been labeled as construct 8. (B) Western blots reflecting the surface presence of N-half and C-half, when co-expressed with cbv1, obtained by surface biotinylation of <i>Xenopus</i> oocytes expressing cbv1+N-half+C-half complexes. Blot image where left and right lanes contain samples from uninjected and N-half+C-half chimera-injected oocytes, respectively. (C) Representative traces of macroscopic current recordings obtained from I/O oocyte membrane patches expressing construct 8; Ca²⁺_i = 10 µM. (D) Averaged G/G_max-V plots from cbv1, cbv1+β1, cbv1+β4, and cbv1+construct 8; Ca²⁺_i = 10 µM. Averaged V_half (E), activation (F) and deactivation (G) time constants (τ_act, τ_deact, respectively) obtained cbv1, cbv1+β1, cbv1+β4, and cbv1+construct 8. *Different from cbv1 (P<0.05); ^#Different from cbv1+β1 (P<0.05). Error bars show SEM; each point represents the average of ≥4 patches.</p

Both TMs of β1 are required for conferring the characteristic phenotype of β1-containing BK channel complexes.

<p>(A) Cartoons depicting the chimeric constructs obtained by swapping TM protein regions between hβ1 and hβ4 subunits. Regions from β1 and β4 are given in black and grey, respectively. (B) Macroscopic current recordings obtained from I/O oocyte membrane patches expressing cbv1+β1TMs₄ (construct 4) and cbv1+β4TMs₁ (construct 5) Ca²⁺_i = 10 µM. (C) Averaged G/G_max-V plots of constructs 1–5 obtained at Ca²⁺_i = 10 µM. Averaged V_half (D), activation (E) and deactivation (F) time constants (τ_act, τ_deact, respectively) from constructs 1–5 obtained at 10 M Ca²⁺_i. *Different from cbv1 (P<0.05); ^#Different from cbv1+β1 (P<0.05). Error bars correspond to SEM; each point represents the average of ≥4 patches.</p

Activation of smooth muscle BK channels by hydrochlorothiazide requires cell integrity and the presence of BK β1-subunits

Thiazide-like diuretics are one of the most commonly used drugs to treat arterial hypertension, with their efficacy being linked to their chronic vasodilatory effects. Previous studies have suggested that activation of the large conductance voltage- and Ca2+-dependent K+ (BK) channel (Slo 1, MaxiK channel) is responsible for the thiazide-induced vasodilatory effect. However, direct electrophysiological evidence supporting this claim is lacking. BK channels can be associated with small accessory β-subunits (β1-β4) that confer specific biophysical and pharmacological characteristics to the current phenotype. The β1-subunit is primarily expressed in smooth muscle cells (SMCs). The effect of hydrochlorothiazide (HCTZ) on BK channel activity was measured using patch-clamp electrophysiology on native SMCs from human umbilical artery (HUASMCs) and HEK293T cells expressing the BK channel (with and without the β1-subunit). HCTZ significantly activated the BK current when evaluated using the whole-cell and cell-attached configurations. However, HCTZ did not affect the unitary conductance and open probability of the BK channel in the inside-out configuration, suggesting an indirect mechanism requiring cell integrity. The increase in BK channel activity due to HCTZ was concentration dependent, with an EC50 of 28 μmol/L, and membrane potential did not influence the concentration relationship. Moreover, our data c learly demonstrated that the HCTZ-induced activation of BK channels required the presence of β1-subunits. A β1-subunit-dependent mechanism that requires SMC integrity leads to HCTZ-induced BK channel activation.Instituto de Estudios Inmunológicos y Fisiopatológico