Differential Functional Contribution of BK Channel Subunits to Aldosterone-Induced Channel Activation in Vascular Smooth Muscle and Eventual Cerebral Artery Dilation

Figures

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  Figure 1. (A) Sketch of a BK channel heterodimer of α (slo1) (orange) and generic β (types 1–4) (green) subunits, with pore in the former between S5 and S6. Four heterodimers make the functional BK channels expressed in most mammalian tissues. The effect of different steroids on BK channel activity is shown, with the number of carbons in the steroid molecule and the BK subunit involved in steroid action in parenthesis. EC: extracellular medium; IC: intracellular medium S: transmembrane segment in α; RCK: regulator of conductance for K+ domain; TM: transmembrane domain in β; DHEA: dehydroepiandrosterone. (B) Structure of aldosterone (C21).

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  Figure 2. Differential scanning fluorometry on isolated BK-associated proteins shows differential binding of aldosterone. (A) Example of normalized traces from a single thermal unfolding run of isolated BK α subunit (slo1; cbv1 isoform). In each run, protein samples were loaded in parallel into multiple capillaries with various aldosterone concentrations. (B) Differences between onset and inflection point of averaged cbv1 curves show significant differences in unfolding at 0.3, 10, and 100 µM aldosterone. Average values reflect data from no fewer than three runs representing biological replicates. (C) Example of normalized traces from a single thermal unfolding run of isolated BK β1 subunit. As in (B), during one run, protein samples were loaded in parallel into multiple capillaries with various aldosterone concentrations. (D) Differences between onset and inflection point of averaged β1 curves show a significant difference at 0.3 and at 100 µM aldosterone. * p < 0.05 unless otherwise stated. Average values reflect data from no fewer than three runs representing biological replicates.

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  Figure 3. Aldosterone activates smooth muscle BK channels from wt mouse middle cerebral artery (MCA) in a concentration-dependent manner; this action is blunted in myocyte BK channels from KCNMB1−/− mice. (A) Representative channel activity traces before (top), during (middle), and after (bottom) application of 3 µM aldosterone to the cytosolic side of inside-out (I/O) patches from KCNMB1−/− myocytes. (B) In these myocytes, channel steady-state activity (NPo) as a function of aldosterone concentration shows a mild increase only at 100 µM. (C) Channel activity traces before (top), during (middle), and after (bottom) application of 3 µM aldosterone to the cytosolic side of I/O patches from wt myocytes. (D) In wt cells, aldosterone action is concentration-dependent: EC50 = 3 µM; ECMAX = 10 µM. In (A,C): red dotted lines and an oblique red arrow indicate the baseline (all channels closed).

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  Figure 4. Aldosterone mildly dilates in vitro pressurized, de-endothelialized middle cerebral arteries (MCA) from wt mice but fails to do so in MCA from KCNMB1−/− mice. (A,B) Representative MCA diameter traces from KCNMB1−/− mouse showing diameter changes in response to aldosterone or depolarizing 60 mM KCl. (C,D) Corresponding results from wt mice show that aldosterone mildly dilates MCA in a concentration-dependent manner.

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  Figure 5. In intact (+endothelium) middle cerebral arteries (MCA) from wt mice, aldosterone-induced dilation is reduced when compared to that in de-endothelialized MCA (Figure 4). (A,B) Representative MCA diameter traces from KCNMB1−/− mouse showing diameter changes in response to aldosterone or depolarizing 60 mM KCl. (C,D) Corresponding results from wt mice show a very small dilation by >0.3 μM aldosterone.