Modulation of the molecular composition of large conductance, Ca²⁺ activated K⁺ channels in vascular smooth muscle during hypertension

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 β1 subunit was decreased relative to the pore-forming α 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 β1 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.

Chronic hypertension increases morbidity and mortality from stroke, coronary artery disease, congestive heart failure, and renal disease (1, 2). Sustained increases in arterial tone are an essential component in the development of hypertension (3, 4). Mounting evidence suggests that sustained depolarization (5, 6) of smooth muscle underlies the increase in arterial tone during hypertension by increasing the open probability of voltage-dependent L-type Ca²⁺ channels (6). The resulting increase in Ca²⁺ influx raises global intracellular Ca²⁺ ([Ca²⁺]_i) and contributes to constriction (7, 8). The molecular mechanisms underlying these changes in vascular smooth muscle function during hypertension are presently unclear.

Unlike a global elevation of [Ca²⁺]_i, highly localized intracellular Ca²⁺ transients (Ca²⁺ sparks), originating from ryanodine-sensitive Ca²⁺-release channels (ryanodine receptors [RyRs]) in the sarcoplasmic reticulum (SR), increase [Ca²⁺]_i locally but do not cause contraction (9). Instead, Ca²⁺ sparks oppose depolarizing contractile stimuli by activating closely apposed, large-conductance, Ca²⁺-activated K⁺ (BK) channels (9, 10). An important implication of this model is that dynamic regulation of BK channel activity by Ca²⁺ sparks may be critical in the determination of vascular tone and blood pressure. Consistent with this, vasoconstrictors, acting through stimulation of PKC, decrease BK channel activity indirectly by inhibiting Ca²⁺ sparks (11). Conversely, increasing Ca²⁺ spark activity through cAMP-dependent signaling or enhancing BK channel coupling to Ca²⁺ sparks causes vasodilation (12, 13). These studies suggest a central role for RyR-BK channel communication via local Ca²⁺ signals in the regulation of arterial tone and blood pressure.

BK channels in vascular smooth muscle are composed of pore-forming α and accessory β1 subunits (14, 15). Coexpression of the β1 subunit results in BK channels with increased Ca²⁺ sensitivity (16, 17). The β1 subunit appears to be uniquely expressed in smooth muscle (18). A functional role for the β1 subunit in vascular smooth muscle was recently demonstrated in genetically engineered mice lacking expression of this subunit (18–20). These studies showed that targeted deletion of the β1 subunit uncoupled BK channels from Ca²⁺ sparks, and thereby decreased the contribution of BK channels to the regulation of vascular tone. Importantly, β1 KO mice were hypertensive (HT), indicating that uncoupling BK channels from RyRs by the loss of the β1 subunit alone is sufficient to induce hypertension.

β1 subunit expression is high in arteries from normotensive (NT) animals (18–20). Consequently, in NT arteries, RyR-BK channel coupling is strong (10). It is unclear, however, whether a disruption of RyR-BK channel communication is involved in the progression from a NT to a HT state. Because proper RyR-BK channel communication is important for normal arterial function, we tested the hypothesis that during hypertension, downregulation of the β1 subunit results in BK channels with a diminished capacity to regulate vascular tone. We found that during Ang II-induced hypertension, BK channels were less sensitive to activation by Ca²⁺ sparks and were less able to regulate vascular tone. In agreement with these observations, we detected a decrease in the expression of the β1 subunit; expression of the α subunit was unchanged. From these observations, we conclude that downregulation of the β1 subunit of the BK channel contributes to vascular dysfunction during hypertension.

Ibtx causes smaller constrictions in HT than NT cerebral arteries. Our first series of experiments examined the functional contribution of BK channel activity to vascular tone in pressurized (60 mmHg) cerebral arteries from NT control (systolic pressure = 124 ± 4 mmHg) and HT rats (systolic pressure = 213 ± 7 mmHg; Figure 1). As shown previously (23, 24), the BK channel-specific inhibitor Ibtx (300 nM) (25) caused a robust constriction of pressurized arteries from NT controls (16% ± 2 %; n = 5 arteries), which is consistent with BK channel activity opposing vasoconstriction. In contrast, pressurized arteries from HT animals constricted weakly to Ibtx (5% ± 2 %; P < 0.05, n = 5 arteries). To test that the diminished capacity of HT arteries to constrict to Ibtx did not reflect an inability of these arteries to constrict, we examined the effects of 60 mM KCl on NT and HT vascular tone. We found that 60 mM KCl evoked significant constrictions of equal magnitude (P < 0.05) in NT (70.8% ± 3.3%; n = 5) and HT (65.6% ± 1.8%; n = 5) (Figure 1c). These data indicate that the weak constrictions induced by Ibtx in HT arteries were not due to these arteries being unable to respond to contractile stimuli. Taken together, our data support the hypothesis that decreased BK channel activity occurs during hypertension.

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.

Uncoupling of Ca²⁺ sparks and BK channels in HT arterial myocytes. One possible mechanism for reducing BK channel activity in HT arteries is a decrease in smooth muscle Ca²⁺ spark activity. Therefore, we examined Ca²⁺ sparks and BK currents in isolated cerebral arterial smooth muscle cells. Figure 2a illustrates representative line-scan confocal images of Ca²⁺ sparks from NT and HT myocytes. The rates of spontaneous Ca²⁺ sparks were similar in NT and HT cells (3.07 ± 0.18 Hz, n = 6 vs. 3.03 ± 0.16 Hz, n = 6; P > 0.05) in voltage-clamped myocytes (holding potential = –40 mV). Interestingly, the amplitude of Ca²⁺ sparks in HT cells was larger than in NT cells (1.63 ± 0.01 F/F₀, n = 707 vs. 1.71 ± 0.01 F/F₀, n = 668; P < 0.05). In addition, Ca²⁺ sparks in HT cells decayed slower than in NT cells; the average time for a Ca²⁺ spark to decay to 50% of its amplitude in HT and NT cells was 91.1 ± 3.9 ms and 63.9 ± 3.18 ms, respectively (P < 0.05, n = 75). These data show that a decrease in Ca²⁺ spark activity cannot account for the decreased BK channel activity observed in HT arteries.

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.

We therefore considered the alternative hypothesis that the efficiency with which Ca²⁺ sparks activate BK currents is diminished during hypertension. To test this, we performed simultaneous measurements of Ca²⁺ sparks and BK currents in control and HT cerebral arterial myocytes (Figure 2b). Measurements were performed at the physiologic holding potential (HP) of –40 mV (8) using the whole-cell patch clamp technique in the perforated-patch configuration. In agreement with previous work (10), Ca²⁺ spark and BK current amplitudes were highly correlated in NT arterial smooth muscle cells. However, note that in HT cells, Ca²⁺ sparks of similar amplitude to those observed in NT cells evoked smaller BK currents (Figure 2c). Indeed, the mean transient BK current amplitude for a given Ca²⁺ spark was approximately 50% lower in HT cells than in control cells (Figure 2d). Interestingly, the duration of BK currents at 50% of their amplitude was similar in HT (20.29 ± 1.00 ms) and NT (21.54 ± 1.02 ms) cells (P > 0.05, n = 100).

It is possible that a reduction in the number of functional BK channels could underlie the smaller BK currents observed in HT myocytes. However, we found that the number of BK channels in excised (inside-out) membrane patches from NT (n = 12) and HT (n = 15) myocytes was similar (about 3 channels per patch, P > 0.05; Figure 3d). In addition, the unitary conductance of BK channels (symmetrical 140 mM K⁺) in NT and HT cells was not different (265 ± 6 picosiemens, n = 5, vs. 268 ± 1 picosiemens, n = 5; P > 0.05). Thus, the smaller BK current amplitudes observed in HT myocytes results from reduced coupling efficiency between Ca²⁺ sparks and BK channels.

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.

Decreased β1 subunit function in HT myocytes. One plausible mechanism for reduced coupling efficiency is a decrease in the sensitivity of BK channels to activation by Ca²⁺. Therefore, we compared the apparent Ca²⁺ sensitivity of BK channels in excised patches from NT and HT myocytes. In these experiments we exposed the intracellular aspect of the BK channels to Ca²⁺ concentrations of 0.1, 1, and 10 μM (HP = –40 mV). Figure 3a shows that at the three Ca²⁺ levels examined, the P_o of BK channels from HT cells was smaller than that of channels from NT cells (P < 0.05, n = 8 patches each). Indeed, at 10 μM Ca²⁺, the P_o of BK channels from HT cells was about 50% smaller than in NT cells.

The pore-forming α subunit of the BK channel possesses intrinsic sensitivity to activation by Ca²⁺ (26). However, the Ca²⁺ sensitivity of these channels is enhanced by the presence of accessory β subunits (27–29). Cerebral arterial smooth muscle cells of β1 KO mice have BK channels with reduced Ca²⁺ sensitivity that are poorly coupled to Ca²⁺ sparks (18, 20). These mice are also HT. Thus, downregulation of the β1 subunit may underlie the changes in BK channel function observed during hypertension. We used functional and molecular approaches to test this hypothesis.

In addition to enhancing Ca²⁺ sensitivity, the β1 subunit modifies gating and pharmacologic features of BK channels. In the presence of the β1 subunit, open dwell times of single BK channels are increased (29). If β1 expression is decreased in HT arterial myocytes, as suggested by the decrease in Ca²⁺ sensitivity, then one would expect that the BK channel open times should be decreased. To test this, we examined the open times of BK channels in NT and HT myocytes by constructing open time histograms at a membrane potential of +40 mV with a free Ca²⁺ of 1 μM. Figure 3b shows representative single-channel recordings and respective open time histograms from NT and HT myocytes. Clearly, the open dwell times of HT BK channels are decreased relative to those of BK channels from NT arteries (9.9 ± 1.5 ms vs. 18.0 ± 2.7 ms, P < 0.05, n = 9 patches each), which is consistent with decreased expression of the β1 subunit of the BK channel in HT arteries.

The β1 subunit also confers sensitivity to acute activation of BK channels by estradiol (30) and the xenoestrogen tamoxifen (31). If the β1 subunit is downregulated as suggested by the observed decrease in BK channel open time and Ca²⁺ sensitivity, then BK channels should be less sensitive to acute activation by tamoxifen (Tam). Therefore, we used Tam as a pharmacologic probe to assess β1 subunit function in NT and HT arterial myocytes. We examined the Tam sensitivity of BK channels in excised patches at a membrane potential of +40 mV with 100-nM free Ca²⁺. Tam exposure (Figure 3c) increased the P_o of BK channels from NT myocytes (4.0 ± 1.2–fold), whereas those from HT myocytes were minimally affected (1.5 ± 0.5–fold; P < 0.05, n = 5 patches each). Reduction of BK single-channel conductance by Tam resides in the pore-forming α subunit (31). Although the effect of Tam on P_o was primarily limited to NT BK channels, Tam produced the same reduction in BK single-channel current amplitude in NT (7.0% ± 0.9 %) and HT (6.8% ± 0.7 %) patches (Figure 3c; P > 0.05, n = 5 each). Thus, the reduced effect of Tam on BK channel P_o in HT patches did not result from the lack of Tam-BK channel interaction. From these results (decreased coupling of BK channels from Ca²⁺ sparks, reduced BK channel Ca²⁺ sensitivity, decreased open dwell time, and decreased sensitivity to activation by Tam), we conclude that, in comparison with NT arteries, β1 subunit function is reduced in HT arterial myocytes.

Downregulation of β1, but not α, mRNA during hypertension. To address the origin of the apparent reduction in β1 subunit function observed in HT arteries, we examined β1 expression at the transcriptional level by conventional RT-PCR. In these experiments, we also examined the expression the pore-forming α subunit, which has been suggested to be upregulated in other models of hypertension (32). Figure 4a demonstrates a clear reduction in the amplification of β1 transcripts with no obvious change in the α signal. To confirm these findings, we used real-time RT-PCR to determine the relative abundance (normalized to 18S RNA) of α and β1 transcripts in NT and HT arteries. Real-time RT-PCR demonstrated that in NT arteries, the relative abundance of α and β1 transcript was equal (Figure 4b; P > 0.05, n = 3 animals). In HT arteries, pore-forming α transcripts were not different from NT arteries (Figure 4b; P > 0.05, n = 3 animals), whereas β1 transcripts were approximately 65% less abundant than in NT arteries (Figure 4b; P < 0.05, n = 3 animals). These results are consistent with the apparent decrease in β1 subunit function and the lack of difference in BK channel density in excised membrane patches (see Figure 3). We conclude that the decrease in BK channel activity observed in HT arteries, including the apparent reduction in β1 subunit function, resulted from downregulation of β1 gene transcripts.

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.

In this study we have presented data supporting the novel hypothesis that during Ang II–induced hypertension, downregulation of the BK channel β1 subunit results in BK channels with reduced open conformation stability and with lower sensitivity to physiologically relevant changes in [Ca²⁺]_i. The implications of our findings are profound. First, changes in the stoichiometric composition of BK channel subunits can occur during pathologic conditions such as hypertension. Indeed, these findings raise the intriguing possibility of differential regulation of BK channel subunit expression as a mechanism for the control of vascular function. Second, decreasing expression of the β1 subunit dramatically reduces the ability of Ca²⁺ sparks to activate BK channels and compromises the ability of the artery to oppose increased contractile stimuli during hypertension. Third, decreased β1 function during hypertension diminishes the possibility of acute modulation of BK channels by estrogen (30).

The mechanisms by which a reduction in BK channel function may lead to increased vasoconstriction have been examined by a series of recent studies. It has been proposed that inhibition of BK channels with Ibtx (23) or inhibition of their physiologic activators, Ca²⁺ sparks (9), results in vascular smooth muscle depolarization thereby increasing the opening of voltage-activated L-type Ca²⁺ channels. The resulting increase in Ca²⁺ influx raises global [Ca²⁺]_i, which causes vasoconstriction and hence contributes to increased arterial pressure. It is important to note that expression of L-type Ca²⁺ channels is reportedly higher in vascular smooth muscle cells of HT than in NT animals (33–35). Thus, membrane depolarization, together with an increase in voltage-activated Ca²⁺ channel number, could conspire to produce a large increase in global [Ca²⁺]_i and vasoconstriction.

This is the first study to examine the role of RyR-BK channel communication and the subunit composition of BK channels in HT vascular smooth muscle. However, the Ca²⁺ sensitivity of single BK channels in smooth muscle of cerebral arteries and the aorta from spontaneously hypertensive rats (SHR), a genetic model of hypertension, has been previously investigated (32, 36). These studies examined the activity of BK channels from SHR and Wistar Kyoto rats (WKY), the animals regularly used as a control for SHR. Smooth muscle cells isolated from these animals were used to record the activity of single BK channels at Ca²⁺ concentrations ranging from 0.1 to 10 μM. In contrast to our findings, these authors (32, 36) found an increased number of BK channels in smooth muscle cells from SHR rats compared with WKY rats. Furthermore, they found no difference in the ability of BK channels to respond to changes in [Ca²⁺]_i. Because hypertension is a heterogeneous polygenic disorder, differences between genetic and acquired models of hypertension are not surprising (37). However, it is important that future studies examine β1 expression in SHR and WKY rats.

At present, the molecular mechanisms underlying downregulation of the β1 subunit in HT rats are unclear. One intriguing possibility is that an increase in Ang II signaling could lead to β1 downregulation. Although Ang II levels in NT and HT subjects may be comparable (38), it is important to note that in humans, an Ang I receptor polymorphism with enhanced responsiveness to Ang II (39) has been associated with essential hypertension (40). Thus, renin-Ang activity may be increased during hypertension even in the absence of elevated Ang II plasma levels. Future experiments will address the nature of the renin-Ang system regulation of β1 expression in vascular smooth muscle.

Our data clearly demonstrate that the molecular composition of BK channels is altered during hypertension. However, the exact subunit stoichiometry of BK channels in HT vascular smooth muscle cells is presently unclear. Comparison of the data shown in Figure 3 with that obtained by Brenner et al. (18) using β1 KO mice may provide some insight. We found that at –40 mV and 10 μM Ca²⁺, the P_o of BK channels in HT cells was about 0.25 (compared with about 0.50 in NT cells). This P_o is significantly larger than the value reported (P_o < 0.01) by Brenner et al. (18) from β1 KO cells under similar experimental conditions. Assuming that mouse and rat BK channels have similar voltage and Ca²⁺ dependencies, our data suggest that not all BK channels in HT smooth muscle cells are devoid of β1. Future experiments will examine the exact subunit stoichiometry of BK channels in NT and HT vascular smooth muscle cells.

We found that Ca²⁺ spark activity was not depressed in smooth muscle cells from HT, Ang II-infused animals. Ca²⁺ spark amplitude and duration were actually greater in isolated HT smooth muscle cells (see Figure 2). However, in vivo activation of PKC by Ang II could inhibit Ca²⁺ sparks, thus decreasing BK channel activity (11). PKC could also inhibit voltage-gated, delayed-rectifier K⁺ channels (41). Thus, increased Ang II signaling could depolarize and thereby constrict vascular smooth muscle by inhibiting Ca²⁺ sparks, reducing β1 expression and/or inhibiting voltage-gated, delayed-rectifier K⁺ currents.

To conclude, the results of this study suggest a novel mechanism underlying smooth muscle dysfunction during hypertension. Our findings raise the intriguing possibility that downregulation of the β1 subunit may be an active component in the natural development of hypertension. Finally, this study indicates that increasing the sensitivity of BK channels to Ca²⁺, either by restoring β1 function or by increasing the intrinsic Ca²⁺ sensitivity of the α subunit, may be a therapeutic approach to correcting vascular dysfunction during hypertension.

We thank Stephen M. Schwartz and Patti Polinsky for help with implanting osmotic minipumps and performing blood pressure measurements. We also thank Rachel Greven for technical assistance and Scott Votaw for help with image analysis. We are also grateful to Bertil Hille and Carmen A. Ufret and Eric G. Chase for critically reading this manuscript. This work was supported by National Institute ofNeurological Disease and Stroke grant NS34905 to L.F. Santana and M.T. Nelson. Support for this study was also provided by the Totman Medical Research Trust Fund and grants HL44455 and HL63722 to M.T. Nelson.

See the related Commentary beginning on page 654.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: Ca²⁺-activated K⁺ (BK); global intracellular Ca²⁺ ([Ca²⁺]_i); ryanodine receptor (RyR); sarcoplasmic reticulum (SR); hypertensive (HT); normotensive (NT); hydroxyethylenediaminetetraacetic acid (HEDTA); open channel probability (P_o); bicarbonate-based PSS (B-PSS); iberiotoxin (Ibtx); holding potential (HP); tamoxifen (Tam); spontaneously hypertensive rats (SHR); Wistar Kyoto rats (WKY).

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