Modulation of the molecular composition of large conductance, Ca(2+) activated K(+) channels in vascular smooth muscle during hypertension

Abstract

Hypertension is a clinical syndrome characterized by increased vascular tone. However, the molecular mechanisms underlying vascular dysfunction during acquired hypertension remain unresolved. Localized intracellular Ca2+ release events through ryanodine receptors (Ca2+ sparks) in the sarcoplasmic reticulum are tightly coupled to the activation of large-conductance, Ca2+-activated K+ (BK) channels to provide a hyperpolarizing influence that opposes vasoconstriction. In this study we tested the hypothesis that a reduction in Ca2+ spark-BK channel coupling underlies vascular smooth muscle dysfunction during acquired hypertension. We found that in hypertension, expression of the beta1 subunit was decreased relative to the pore-forming alpha subunit of the BK channel. Consequently, the BK channels were functionally uncoupled from Ca2+ sparks. Consistent with this, the contribution of BK channels to vascular tone was reduced during hypertension. We conclude that downregulation of the beta1 subunit of the BK channel contributes to vascular dysfunction in hypertension. These results support the novel concept that changes in BK channel subunit composition regulate arterial smooth muscle function.

Figure 1

HT cerebral arteries are less sensitive to Ibtx than NT cerebral arteries. (a) Representative arterial diameter records from pressurized (60 mmHg) NT (black) and HT (red) arteries before and after the application of Ibtx (300 nM). Before the addition of Ibtx, the diameter of the HT and NT pressurized arteries were, respectively, 87 μm (passive diameter = 146 μm) and 130 μm (passive diameter = 148 μm). (b) Mean ± SEM of Ibtx-induced constriction in NT and HT arteries. (c) Mean ± SEM of KCl-induced (60 mM) constriction in NT and HT arteries. *P < 0.05.

Figure 2

Reduced coupling between Ca²⁺ sparks and BK channels in HT arterial myocytes. (a) Representative line-scan images of Ca²⁺ sparks from NT and HT myocytes (left side). The traces to the right show the time course of [Ca²⁺]_i in the regions of the images delimited by the bars located at the end of each line-scan image. (b) Simultaneous BK current (top; HP = –40 mV) and Ca²⁺ sparks (bottom) recordings from NT and HT myocytes. In all cases, Ca²⁺ sparks had an associated BK current. However, on occasion, a Ca²⁺ spark outside the imaged area would evoke a BK current (e.g., the fifth BK current from left in NT cell). Dashed lines indicate the mean pA or F/F₀ (as appropriate) for each representative trace. (c) Relationship between BK current and Ca²⁺ spark amplitudes in NT (circles; 46 sparks from 6 cells) and HT (triangles; 41 sparks from 6 cells) myocytes. Data for this plot were obtained from traces similar to those shown in b. The smooth lines represent the best linear regression fits using a least-squares routine. The slope of the line used to fit the NT and HT data was, respectively, 112.4 ± 26.8 and 43.2 ± 9.2 pA/Ca²⁺ (F/F₀). (d) Coupling strength (BK current amplitude divided by Ca²⁺ spark amplitude) in NT and HT myocytes. *P < 0.05.

Figure 3

Functional and pharmacologic properties of single BK channels indicate decreased β1 subunit function in HT myocytes. (a) Ca²⁺sensitivity of BK channels in inside-out patches (HP = –40 mV) from NT and HT myocytes. Shown to the left are representative single BK channel records taken from NT and HT patches in the presence of 1 or 10 μM Ca²⁺. The bar plot to the right shows the mean ± SEM P_o of BK channels in NT and HT patches at three Ca²⁺ concentrations. (b) Open-time analysis of BK channels in inside-out patches from NT and HT myocytes. Shown to the left are representative single BK channel records taken from NT and HT patches at +40 mV in the presence of 1 μM Ca²⁺. The open-time histograms of these BK channels from NT and HT myocytes are shown in the center. Histograms were fitted with a single exponential function. The bar plot to the right shows the mean ± SEM τ_open of BK channels in NT and HT cells. (c) Tam (1 μM) sensitivity of BK channels in inside-out patches (HP = +40 mV; 100 nM free Ca²⁺) from NT and HT myocytes. Shown to the left are representative single BK channel records taken from NT and NT before and after the application of Tam. The bar plot to the right shows the mean ± SEM fold change in the P_o of BK channels in NT and HT cells after the application of Tam. (d) Number of BK channels per patch. Dashed lines indicate open channels. o, open channel; c, closed channel. *P < 0.05.

Figure 4

The mRNA levels of β1, but not α, are downregulated in HT cerebral arteries. (a) Conventional RT-PCR analysis of α and β1 transcript expression in NT and HT arteries. STD = 100-bp marker; NEG = nontemplate control. (b) Bar plot of the α and β1 transcript abundance in NT and HT smooth muscle as determined by real-time RT-PCR. For each sample, α and β1 amplifications were normalized to the amount of 18S RNA present. See the Methods section for all primer and probe sequences. *P < 0.05.