large-conductance Ca2+-activated K+ channel
Dulbecco's modified Eagle's medium
- NP o
normalized open probability
physiological saline solution
spontaneous transient outward current
vascular smooth muscle cell
- V m
The plasma membrane large-conductance Ca2+-activated, K+ channel (BKCa) is a major ion channel contributing to the regulation of membrane potential.
Activation of large-conductance Ca2+-activated K+ channel by both depolarization and increased intracellular Ca2+ results in hyperpolarization that acts to limit agonist and mechanically induced vasoconstriction in small arteries.
Using patch-clamp techniques we demonstrate that regional differences exist in how BKCa is regulated, particularly with respect to its Ca2+ sensitivity.
Using single-channel recordings and siRNA to manipulate protein subunit expression, it is argued that the β1-subunit plays a more dominant role in cerebral blood vessels as compared with small arteries from skeletal muscle.
Subtle differences in the regulation of membrane potential in different vascular beds allow local blood flow and pressure to be closely adapted to the tissue's metabolic needs.
Abstract β1-Subunits enhance the gating properties of large-conductance Ca2+-activated K+ channels (BKCa) formed by α-subunits. In arterial vascular smooth muscle cells (VSMCs), β1-subunits are vital in coupling SR-generated Ca2+ sparks to BKCa activation, affecting contractility and blood pressure. Studies in cremaster and cerebral VSMCs show heterogeneity of BKCa activity due to apparent differences in the functional β1-subunit:α-subunit ratio. To define these differences, studies were conducted at the single-channel level while siRNA was used to manipulate specific subunit expression. β1 modulation of the α-subunit Ca2+ sensitivity was studied using patch-clamp techniques. BKCa channel normalized open probability (NPo) versus membrane potential (Vm) curves were more left-shifted in cerebral versus cremaster VSMCs as cytoplasmic Ca2+ was raised from 0.5 to 100 μm. Calculated V1/2 values of channel activation decreased from 72.0 ± 6.1 at 0.5 μm Ca2+i to −89 ± 9 mV at 100 μm Ca2+i in cerebral compared with 101 ± 10 to −63 ± 7 mV in cremaster VSMCs. Cremaster BKCa channels thus demonstrated an ∼2.5-fold weaker apparent Ca2+ sensitivity such that at a value of Vm of −30 mV, a mean value of [Ca2+]i of 39 μm was required to open half of the channels in cremaster versus 16 μm[Ca2+]i in cerebral VSMCs. Further, shortened mean open and longer mean closed times were evident in BKCa channel events from cremaster VSMCs at either −30 or 30 mV at any given [Ca2+]. β1-Subunit-directed siRNA decreased both the apparent Ca2+ sensitivity of BKCa in cerebral VSMCs and the appearance of spontaneous transient outward currents. The data are consistent with a higher ratio of β1-subunit:α-subunit of BKCa channels in cerebral compared with cremaster VSMCs. Functionally, this leads both to higher Ca2+ sensitivity and NPo for BKCa channels in the cerebral vasculature relative to that of skeletal muscle.
The large-conductance Ca2+-activated K+ channel (BKCa) has been demonstrated to be a major ion channel involved in the control of membrane potential in vascular smooth muscle cells (VSMCs; Nelson & Quayle, 1995). Through this action it is viewed as a major determinant of tone, or contractile activity, in arterial smooth muscle. Specifically, BKCa is activated by a number of depolarizing stimuli and/or changes in intracellular calcium, including effects produced by contractile agonists and intraluminal pressure, to cause an opposing hyperpolarization and thereby limit the extent of constriction (Nelson & Quayle, 1995).
In VSMCs, BKCa is comprised of a tetramer of α-subunits that co-assemble around a central axis to form a single K+-selective conduction pore. The addition of up to four accessory β-subunits (typically β1 in VSMCs) modifies the conduction properties, particularly increasing the channel's sensitivity to intracellular Ca2+ (McManus et al. 1995; Dworetzky et al. 1996; Bao & Cox, 2005), although the presence of the β1-subunit may also contribute to kinetics of channel gating, responsiveness to pharmacological modulators and trafficking of channels to the plasma membrane (Valverde et al. 1999; Dick et al. 2001; Dick & Sanders, 2001; Ledoux et al. 2006; Toro et al. 2006; Kim et al. 2007; Zarei et al. 2007; Hill et al. 2010).
Regulation of BKCa function in VSMCs is complex and occurs at multiple levels (Nelson & Quayle, 1995; Ledoux et al. 2006; Hill et al. 2010). Major regulatory mechanisms include changes in channel composition through expression of splice variants and alternate regulatory subunits; modulation by voltage, Ca2+, protein kinases and a number of small molecules; and cellular architecture (Schubert & Nelson, 2001; Shipston, 2001; Ledoux et al. 2006). As a result of these factors, it has been considered likely that BKCa may exhibit considerable regional/tissue-specific heterogeneity (Hill et al. 2010).
In a recent study (Yang et al. 2009), we presented data suggesting that arteriolar smooth muscle cells from small cerebral arteries and cremaster muscle arterioles may differ in the subunit composition of BKCa. Specifically, cerebral artery smooth muscle cells were described as having a higher β1-subunit:α-subunit ratio, which would be consistent with a greater apparent Ca2+ sensitivity. These observations were based on whole-cell recordings of K+ currents, responsiveness to pharmacological agents (17-β-oestradiol (E2) and 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl) phenyl]- 5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619)) reported to exhibit subunit-specific effects with respect to modulation of BKCa (Gribkoff et al. 1996; Valverde et al. 1999; Dick & Sanders, 2001; De Wet et al. 2006), and measurements of protein and mRNA expression for both the α- and β-subunits of BKCa. Due to factors such as the large single-channel conductance of BKCa and possible variation in the stoichiometry of the holo-channel (complete α- and β-subunit complex), these approaches are indirect and do not necessarily account for functional variation due to differences in channel number. Indeed, in the earlier study (Yang et al. 2009), differences in the overall expression of channel subunit protein were described as well as changes in the β1-subunit:α-subunit ratio.
On the basis of the above information, the present studies took two approaches to further examine differences in BKCa function in VSMCs from cerebral and cremaster muscle small arteries. Firstly, electrophysiological properties of BKCa were examined at the single-channel level, thereby allowing differences in voltage and Ca2+ sensitivity to be determined more precisely. Secondly, subunit-specific siRNA was used, in vitro, to manipulate the expression of the channel. The aim of these latter studies was to determine whether the biophysical phenotype of BKCa in cerebral VSMCs could be altered to more closely resemble that of cremaster VSMCs. From a physiological perspective it is suggested that regional variation in the regulation of BKCa may provide a mechanism for precise regulation of vascular tone in accordance with local metabolic demands. Consistent with this, our earlier studies have shown differences in the relationship between Vm and myogenic tone in cerebral and cremaster small arteries as well as in the apparent function of BKCa (Kotecha & Hill, 2005; Yang et al. 2009).
Studies used male Sprague–Dawley rats weighing 180–280 g. Prior to use, animals were housed in a temperature-, humidity- and light-controlled animal facility with free access to standard rat chow and drinking water. All procedures and protocols were approved by the Animal Care and Use Committee of the University of Missouri (USA). Rats were anaesthetized with sodium pentobarbital (Nembutal, 100 mg kg body weight−1) given by intraperitoneal injection. Cremaster muscles were surgically removed, as previously described (Meininger et al. 1991; Hill et al. 2000), and placed in a cooled (4°C) dissection chamber. Following death by anaesthetic overdose, a craniotomy was performed, and the brain removed and similarly placed in a cooled dissection chamber.
Vessel isolation and preparation of cells
VSMCs from first- and second-order arterioles (1A/2A) were isolated as previously described (Yang et al. 2009). In brief, cremaster muscles were excised and pinned flat for vessel dissection at 4°C in Ca2+-free physiological saline solution (PSS) containing (in mm): NaCl, 140; KCl, 5.6; MgCl2, 1.0; NaH2PO4, 1.2; d-glucose, 5.0; sodium pyruvate, 2.0; EDTA, 0.02; Mops, 3; plus 0.1 mg ml−1 BSA (USB Corporation, Cleveland, OH, USA). Dissected segments of arterioles were transferred to a 1 ml tube of low-Ca2+ PSS containing (in mm): NaCl, 144; KCl, 5.6; CaCl2, 0.1; MgCl2, 1.0; Na2HPO4, 0.42; Hepes, 10; sodium pyruvate, 2; and 1 mg ml−1 BSA at room temperature (RT) for 10 min. The solution was decanted and replaced with a similar solution containing 26 U ml−1 papain and 1 mg ml−1 dithiothreitol (DTT). The vessels were incubated for 30 min at 37°C with occasional agitation, then transferred to a new tube containing low-Ca2+ PSS containing 1.95 U ml−1 collagenase (Type H FALGPA), 1 mg ml−1 soybean trypsin inhibitor and 75 U ml−1 elastase, and incubated for 7–10 min at 37°C. After further digestion, the remaining fragments were gently rinsed two–three times with low-Ca2+ PSS and gently triturated using a fire-polished Pasteur pipette to release single cells. Spindle-shaped arteriolar myocytes were used within approximately 4 h of isolation. Smooth muscle cells from rat cerebral arteries were enzymatically isolated as previously described (Wu et al. 2007). Briefly, arterial segments were placed in low-Ca2+ solution as above (37°C, 10 min). Vessels were then exposed to a two-step digestion process that involved: (1) 15 min incubation in isolation media (37°C) containing 0.6 mg ml−1 papain and 1.8 mg ml−1 DTT; and (2) 5–6 min incubation in low-Ca2+ PSS with 0.7 mg ml−1 type F collagenase and 0.4 mg ml−1 type H collagenase. Following enzyme treatment, tissues were washed repeatedly with ice-cold low-Ca2+ PSS and triturated with a fire-polished pipette. Isolated VSMCs were stored in ice-cold isolation medium for use within 4 h.
The rationale for using different enzymatic procedures for isolating cremaster and cerebral arterial myocytes was based on methods described in the literature for these specific vessels and differences in connective tissue/adventitia (Clifford et al. 2011). Control experiments have previously been performed to ensure that the procedures, per se, did not introduce differences in electrophysiological and Ca2+ handling properties (Yang et al. 2009).
In vitro siRNA-mediated knockdown of α- and β-subunits of BKCa
Segments of small cerebral arteries and cremaster muscle arterioles were dissected as described. Either siRNA-directed α-subunit (Qiagen, Valencia, CA, USA; Catalogue number SI01528233) or β1-subunit (Santa Cruz, Santa Cruz, CA, USA; Catalogue number SC155999) of rat BKCa was introduced into the vessel segments using either the transfection agent FuGene6 (Roche, Diagnostics, IN, USA) or reverse permeabilization (Raina et al. 2008). As a control for potential off-target effects of the transfection procedure, additional experiments were performed using an unrelated siRNA (negative control; Qiagen Catalogue number 1022563).
In brief, 8 μl of FuGene6 (Roche) was mixed with 92 μl DMEM/F12 medium (without serum) for 5 min at RT, then 3 μg siRNA (100–150 nm) was added and mixed for 45 min. Isolated vessels were transferred to the DMEM/F12 medium containing siRNA for 3 h (RT), after which the medium was changed to DMEM/F12 containing 15 mm Hepes, 1 mm l-glutamine, 50 U ml−1 penicillin, 50 μg ml−1 streptomycin, and the appropriate siRNA was added and maintained in an incubator (37°C) for 48 h. For reversible permeabilization, isolated arteries were transferred to culture dishes and exposed to three successive solutions (4°C) containing (in mm): (1) EGTA, 10; KCl, 120; ATP, 5; MgCl2, 2; Tes, 20 (pH 6.8; 20 min); (2) EGTA, 0.1; KCl, 120; ATP, 5; MgCl2, 2; Tes, 20; siRNA, 100 nm (pH 6.8; 3 h); and (3) EGTA, 0.1; KCl, 120; ATP, 5; MgCl2, 10; Tes, 20; siRNA, 100 nm (pH 6.8; 20 min). Subsequently, vessels were bathed in a fourth solution containing (in mm): NaCl, 140; KCl, 5; MgCl2, 10; glucose, 5; Mops, 2 (pH 7.1, 22°C), in which [Ca2+] was gradually increased from 0.01 to 0.1 to 1.8 mm every 15 min. Vessel segments were then placed in non-serum DMEM/F12 culture medium (supplemented with 15 mm Hepes, 2 mm sodium pyruvate, 1 mm l-glutamine, 50 U ml−1 penicillin and 50 μg ml−1 streptomycin) and maintained in an incubator (37°C) for 48 h. At the end of the 48 h incubation period, cells were enzymatically isolated for patch-clamp studies as described above. An additional set of cerebral vessels (supracerebellar arteries) subject to reverse permeabilization and either an α-subunit-directed siRNA or the control siRNA were used to demonstrate the effect of decreasing channel expression on vessel myogenic responsiveness.
To confirm uptake of siRNA into VSMCs using the transfection procedures, additional experiments were performed using a fluorescent control RNA sequence (ex 488 nm; Qiagen). In single-channel studies the β1-subunit-directed siRNA pool was labelled with the fluorescent label, fluorescein isothiocyanate (FITC; ex 488 nm; Qiagen). Following washout of the FuGene6 reagent, vessel segments were imaged using confocal microscopy. Knockdown of the α-subunit was confirmed using Western blotting.
Whole-cell patch-clamping for measurement of outward currents and spontaneous transient outward currents (STOCs)
Conventional patch-clamp (Hamill et al. 1981) electrophysiology and protocols were used to measure macroscopic whole-cell potassium currents (IK). An amplifier (EPC-10, HEKA, Germany) was controlled by a Dell computer using Patchmaster and Igor Pro 6.11 (Wavemetrics, Inc., Lake Oswego, OR, USA) used for data analysis. Micropipettes were pulled from borosilicate glass tubing (Corning 8161; i.d. 1.2 mm; o.d. 1.5 mm; Warner Instruments Corp., Hamden, CT, USA) using a Sutter P-97 electrode puller (Sutter Instrument Co., Novato, CA, USA). Pipette tip resistances ranged from 3.0 to 5.0 MΩ when filled with standard intracellular solution. Whole-cell K+ currents were evoked by voltage steps delivered from a typical holding potential of −60 mV to potentials ranging from −70 to +70 mV, in 20 mV increments. Cell capacitance ranged between 14 and 17 pF, and was measured with the cancellation circuitry in the voltage-clamp amplifier. The series resistance (<10 MΩ) was compensated to minimize the duration of the capacitive surge. Subtraction of leak currents was not performed. Whole-cell currents were normalized to cell capacitance and expressed as picoampere per picofarad (pA pF−1). The presence of BKCa currents was confirmed by the existence of an iberiotoxin (IBTX)-sensitive component of total K+ current. All experiments were performed at RT. For whole-cell recordings, the bath solution contained (in mm): NaCl, 140; KCl, 5.4; CaCl2, 1.5; MgCl2, 1; glucose, 10; Hepes, 10; sodium pyruvate, 2 (pH 7.4). The 140 mm K+ pipette solution contained (in mm): KCl, 140; NaCl, 8; EGTA, 1–2; Mg-ATP, 3; Hepes, 10 (pH 7.2); CaCl2 was added to bring free [Ca2+] to 100–400 nm. Mg-ATP was included to inhibit ATP-sensitive K+ channels and provide substrate for energy-dependent processes. Where addition of other reagents to the bath and/or pipette solutions was required, details are given in specific protocols and figure legends.
STOCs were recorded at a holding potential of 20 mV as previously described (Yang et al. 2009). For determining the frequency of STOCs, a 10pA threshold amplitude was applied for detection.
Single-channel recording and analysis
Unitary BKCa channel currents in single SMCs from both cremaster muscle arterioles and cerebral arteries were recorded using the inside-out patch-clamp configuration (Hamill et al. 1981). Cells were superfused in a chamber (volume 0.5–1 ml) at 1 ml min−1 (ValveLink 8.2; Automate Scientific; solution exchanges were complete within 30–60 s). The resistance of a pipette filled by pipette solution was 8–12 MΩ and the typical seal resistance was >10 GΩ. Currents were amplified using an EPC-10 patch-clamp amplifier, sampled at 2–5 kHz and filtered at 2.9 kHz via a four-pole low-pass Bessel filter. Data acquisition and analysis were performed using Patchmaster 2.3 (HEKA), Igor 6.11 (for graphs and curve fitting), TAC 4.1 (Bruxton Inc, Seatle, WA, USA; for determination of normalized open probability (NPo)), and a custom software program developed by Sohma and Kubokawa (Kubokawa et al. 2005) for calculation of open and shut times.
All experiments were performed at RT. Pipette solution for inside-out patch-clamp studies contained (in mm): KCl, 140.0; CaCl2, 1.0; MgCl2, 1.0; Hepes, 10.0; EGTA, 1.0; adjusted to pH 7.4 with KOH. The bath solution contained (in mm): KCl, 140.0; EGTA, 2.0; Hepes, 10.0; adjusted to pH 7.2 with KOH. Various amounts of 0.1 m CaCl2 were added to give the desired concentrations of free Ca2+ (10−9 to 10−6 m). The solution without added CaCl2 is referred to as 0 Ca2+. Free Ca2+ concentrations of 10, 30, 100 and 1000 μm were made by directly adding CaCl2 into the bath solution without EGTA. The levels of free Ca2+ in the solutions were confirmed using a calcium electrode (Orion Model 93–20; WPI, Sarasota, FL, USA).
Unitary BKCa channel amplitude was determined from amplitude histograms fitted with a Gaussian function. The unitary conductance (γ) was determined from the slope of a least-squares linear fit of the unitary current amplitude–voltage (I–V) relationship. The probability of a single channel being open (Po) was calculated from the mean channel open probability using the following equation.
where N is the number of maximally activated channels with a [Ca2+]i of 100 μm, n is the number of channels observed at the same time, and tn is the probability that n channels are simultaneously open, which was obtained by fitting the amplitude histogram with a Gaussian function (Kubokawa et al. 2005).
P o and Vm data obtained from inside-out patches at each [Ca2+]i were fitted using a Boltzmann function as described below, using a least-squares regression analysis (e.g. Sohma et al. 1994).
where Pmax is the maximal Po, Vm is the membrane potential tested, V1/2 is the membrane potential at which Po is half-maximal, and z is the equivalent number of charges moving across the transmembrane potential during an open–close transition (the gating charge or Boltzmann constant). F, R and T are the Faraday constant, the gas constant and the temperature, respectively.
The Po and [Ca2+]i data at a constant Vm obtained from inside-out patches were best fitted using the Hill equation described below.
where KCa and NCa are the dissociation constant and the Hill coefficient for Ca2+, respectively.
Mean channel open and shut times were calculated from recordings of single channels that were maintained for >5 min and fitted by the following equations:
where max is the maximal opening time (or shut time) and half is the half open (or shut) time.
After 48 h exposure to a specific siRNA or control sequence, cerebral artery segments were homogenized using a cooled pestle and mortar containing cold RIPA buffer plus 1% protease inhibitor cocktail (Sigma; total volume 25 μl). The homogenates were transferred to micro-centrifuge tubes, incubated on ice for 30 min and sonicated (45 s). Homogenates were then centrifuged at 6000 g for 1 min. The supernatants were removed and total protein concentration determined using the BCA protein assay kit (Pierce, ThermoFisher, Rockpoint, IL, USA).
Aliquots containing equal amounts of total protein were mixed with 1× Laemmli sample buffer containing β-mercaptoethanol (5%) and heated at 95°C for 5 min. α-Actin content of each sample was determined in parallel to normalize relative expression to total protein. Detection and quantification of BKCaα-subunit was performed using a sensitive three-step Western blot method, as described by Johnson et al. (2009). α-Subunits were detected using a polyclonal rabbit anti-KCa (Alomone; APC-107 at 1:200 dilution) in combination with goat anti-rabbit IgG, biotin-SP conjugate (Millipore; AP132B) at 1:20,000 dilution, and poly-HRP streptavidin (Pierce No. N200) at 1:100,000. Smooth muscle α-actin was detected using monoclonal anti-α smooth muscle actin primary antibody (Sigma A5228; 1:2000 dilution) and anti-mouse IgG (whole-molecule)-peroxidase-conjugated secondary antibody produced in rabbit-IgG (Sigma A9044; 1:5000 dilution). Chemiluminescence and quantification of band intensities were performed using a Bio-Rad ChemiDoc XRS+ system.
Assessment of vessel function in cannulated arteries
Isolated and cannulated arterioles were studied as previously described (Raina et al. 2008; Yang et al. 2009). In brief, approximately 48 h after reverse permeabilization and exposure to siRNA, artery segments were cannulated on glass micropipettes and allowed to develop spontaneous myogenic tone in the absence of intraluminal flow. Pressure-diameter relationships were then determined under active (Kreb's buffer superfusion containing 2.0 mm Ca2+) and passive (Kreb's buffer superfusion containing 0 mm Ca2+ plus 2 mm EGTA) conditions. Diameter measurements were taken at intraluminal pressures of 10, 30, 50, 70 and 90 mmHg using video microscopy. For data presentation, active diameter measurements for each vessel were normalized to their individual passive diameters at 70 mmHg (normalized diameter = active diameter/passive diameter × 100 at 70 mmHg).
Chemicals and solutions
Unless stated, general chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA). Papain, soybean trypsin inhibitor and collagenase (Type F and H) were purchased from Sigma-Aldrich, and elastase from Calbiochem (Enzo, La Jolla, CA, USA). NS-1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl) phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one) was obtained from Biomol International, L.P. (Plymouth Meeting, PA, USA). E2 and IBTX were purchased from Sigma-Aldrich. NS-1619 stock (10 mm) was made with DMSO, while a stock solution of E2 (1 mg ml−1) was made in ethanol. IBTX was dissolved in distilled water.
Summary data are expressed as mean ± SEM. Statistical significance was determined using Student's t test for paired or unpaired data when two groups were compared. Where comparisons involved three or more groups, ANOVA was used. Significance levels of P < 0.05 were considered significant.
Single-channel characteristics of BKCa in VSMCs from small arteries of cremaster and cerebral circulations
Example recordings of BKCa currents for cremaster and cerebral VSMCs are shown in Fig. 1A and B, respectively. Averaged I–V curves were constructed (Fig. 1C) from the group data, and unitary BKCa channel amplitude determined at each membrane potential from amplitude histograms that were fitted with a Gaussian function. Under symmetrical K+ conditions (140 mm) the unitary conductance (γ) of BKCa channel was calculated to be similar in both VSMC preparations: 238 ± 5 and 247 ± 3 pS for cremaster and cerebral VSMCs, respectively. These values are consistent with previously reported conductance levels for BKCa (Nelson & Quayle, 1995; Jackson & Blair, 1998; Wei et al. 2005), and suggest that the same basic ion channel was being compared between vascular beds. Further, the selective BKCa inhibitor, IBTX (10−7 m) similarly inhibits K+ currents in both VSMC preparations (Yang et al. 2009).
As BKCa activity is known to be dependent on the prevailing Vm and Ca2+ levels, initial studies compared the relative sensitivity to these variables in cremaster and cerebral VSMCs. Using inside-out patches, recordings were performed over an Vm range of −80 to +80 mV at bath [Ca2+] ranging from 0.5 to 100 μm (example recordings at −40 mV are shown in Fig. 2A and B). Plotting NPo vs. Vm showed BKCa channel open probability in cerebral VSMCs to be more sensitive to [Ca2+]i than that of cremaster VSMCs, such that for each Ca2+ concentration tested, the NPo vs. Vm curves were significantly left-shifted (Fig. 2C).
Table 1 shows the calculated Boltzmann constants (Z) for the curves fitted to the data presented in Fig. 2C. No significant differences (P > 0.05) in the values of Z were observed between cremaster and cerebral VSMCs at any given Ca2+ level, consistent with a similar voltage sensitivity for BKCa in both cell types. To determine relative Ca2+ set point values (Carl et al. 1996; Jackson & Blair, 1998; Lin et al. 2003), the half-maximal voltages for channel activation (i.e. V1/2 in mV) were derived from the curves displayed in Fig. 2C and plotted as a semi-log function against free [Ca2+] (μm; Table 2 and Fig. 2D). From the plotted linear relationships, at V1/2= 0 mV there was a requirement for 5.0 μm[Ca2+] in cerebral VSMCs compared with 12.1 μm in cremaster cells. An alternative view of the data, as illustrated in Table 2, is that at comparable Ca2+ levels, cremaster VSMCs require an additional depolarization of approximately 26 mV to overcome their relative decrease in Ca2+ sensitivity and exhibit similar levels of channel opening.
|[Ca2+]i (μm)||Z Cremaster VSMC||Z Cerebral VSMC|
|0.5||2.40 ± 0.31||1.87 ± 0.21|
|1.0||1.88 ± 0.22||1.70 ± 0.20|
|3.0||1.90 ± 0.32||1.80 ± 0.23|
|10||1.88 ± 0.21||1.70 ± 0.18|
|30||1.55 ± 0.25||1.60 ± 0.18|
|100||1.10 ± 0.12||0.92 ± 0.08|
|[Ca2+]i (μm)||Cremaster mean V1/2 (mV)||Cerebral mean V1/2 (mV)||ΔV1/2 (mV)†|
|0.5||101.0 ± 9.8||72.0 ± 6.1*||29.0|
|1.0||56.8 ± 8.9||33.5 ± 5.6*||23.3|
|3.0||45.6 ± 5.9||20.3 ± 3.6*||25.3|
|10||11.1 ± 3.6||−17.1 ± 2.6*||28.9|
|30||−27.6 ± 4.2||−49.2 ± 7.2*||21.6|
|100||−63.0 ± 6.6||−89.5 ± 8.9*||26.5|
|25.8 ± 1.3|
To further contrast differences in the biophysical properties of BKCa from cremaster and cerebral VSMCs, mean open and shut times were calculated at −30 and +30 mV over a [Ca2+] range of 0.3–30 μm. These analyses were limited to a subset of cells where recordings were maintained for more than 5 min and patches contained an apparent single channel. Figure 3 illustrates that at both holding potentials, cerebral VSMCs showed significantly longer open times (Fig. 3A) and shorter closed times (Fig. 3B) at all [Ca2+]. Data for mean open time vs.[Ca2+] (at holding potentials of both −30 and +30 mV) showed a shallow curvilinear relationship, although the general slope of the relationship appeared steeper for the cerebral cells. Closed time vs.[Ca2+] data demonstrated definite curvilinear relationships consistent with the possible existence of multiple closed states (not further examined in this study).
Manipulation of BKCa phenotype with subunit-directed siRNA: whole-cell studies
siRNA directed to either the α- or β1-subunit of BKCa was administered to isolated cremaster arterioles or cerebral small arteries using FuGene6, as described in the Methods. In some studies reverse permeabilization was used as an alternate transfection approach with similar results (data not shown). Uptake of siRNA was confirmed by confocal microscopy using fluorescent siRNA species (both an unrelated FITC-siRNA and a specific FITC-labelled siRNA directed at the β1-subunit of BKCa; Fig. 4A and B). Western blotting further confirmed that the siRNA treatment was successful in decreasing protein content at 48 h (Fig. 4C). Thus, in cerebral vessels siRNA significantly decreased αBKCa protein to 0.13 ± 0.02 compared with 0.25 ± 0.08 (density relative to actin; n= 4, P < 0.05) in vessels treated with an unrelated control siRNA; this indicated an effective decrease in protein expression of approximately 50%.
As a further indicator of efficacy of the siRNA approach, myogenic responsiveness was determined in cannulated cerebral arteries treated with the α-subunit-directed siRNA. Consistent with a decrease in K+ channel activity, vessels exposed to the specific siRNA showed increased myogenic tone relative to vessels treated with the unrelated control siRNA preparation (Fig. 4D). For example, at an intraluminal pressure of 70 mmHg α- siRNA-treated vessels had a normalized diameter (D/D70passive%) of 48.7 ± 4.5 compared with 64.0 ± 2.6 in control siRNA-treated vessels (P < 0.05). No differences were observed in the passive pressure–diameter relationships for the two vessel sets.
To examine the functional consequences of the subunit-specific siRNA exposure, cells were dispersed from vessels after 48 h of siRNA treatment and whole-cell recordings were performed. Initial studies examined the utility of the approach by examining the effects of siRNA directed toward the α (pore forming)-subunit. Consistent with a reduction in the protein, and hence the functional channel, α-subunit siRNA decreased K+ current in both cremaster ( Fig. 5A) and cerebral (Fig. 5B) VSMCs. In addition to decreasing basal K+ current, α-subunit siRNA decreased responsiveness to the pharmacological BKCa activators NS-1619 and E2 (Fig. 5C and D). Previous studies have reported NS-1619 to exert its BKCa enhancing effects predominantly through the α-subunit (Gribkoff et al. 1996), while E2 acts on the β-subunit to increase activity of the holo-channel (Valverde et al. 1999; Dick & Sanders, 2001; De Wet et al. 2006). As the α-subunit-directed siRNA decreases the amount of functional channel, a decrease in responsiveness to both agents was predicted.
Effects of β1-subunit-directed siRNA on cremaster and cerebral VSMC BKCa function are shown in Fig. 6A and B, respectively. Relative to the control siRNA treatment, β1-subunit siRNA caused only a slight reduction in total K+ current and responsiveness to NS-1619 in cremaster and cerebral VSMCs. The β1-subunit siRNA did, however, cause a significant reduction in responsiveness to E2 in both VSMC preparations (Fig. 6A and B), consistent with an effect on the modulatory β-subunit of the BKCa channel in VSM.
As an additional index of BKCa function, STOCs were recorded. The effects of α- and β1-subunit-directed siRNA on the characteristics of STOCs are shown in Fig. 7. STOC frequency and amplitude were reduced in both cell preparations, with the magnitude of inhibition being most dramatic in the cerebral VSMCs (Fig. 7B–D). Specifically, α-subunit siRNA caused a marked reduction in both STOC frequency and amplitude in cremaster and cerebral VSMCs. β1-Subunit siRNA treatment also caused reductions in STOC frequency and amplitude; however, the relative decrease in STOC amplitude appeared much greater in cerebral (0.32 ± 0.03 relative to control) compared with cremaster VSMCs (0.86 ± 0.04 relative to control; Fig. 7D).
Manipulation of BKCa phenotype with subunit-directed siRNA: single-channel recordings
To further study the effects of β1-subunit-directed siRNA on cell phenotype, additional studies were performed at the single-channel level using cerebral VSMCs. To ensure only cells receiving siRNA were studied, experiments utilized a FITC-labelled β1-subunit siRNA and fluorescence microscopy. Example tracings (recorded at both −30 and +30 mV) showing that β1-subunit-directed siRNA decreased single-channel opening are displayed in Fig. 8A. Results are shown compared with cerebral VSMCs similarly treated with an unrelated control siRNA. Channel opening (NPo) was further shown to be less in β1 siRNA-treated cells compared with the control siRNA across the [Ca2+] range studied, such that there was a significant rightward shift in the NPo free Ca2+ relationship at holding potentials of both −30 and +30 mV (Fig. 8B). Figure 8C displays (all curves plotted with maximum response = 1) NPo data at varying [Ca2+] obtained for β1 and control siRNA-manipulated cerebral VSMCs; Fig. 8D shows NPo vs. varying [Ca2+] data for BKCa activity in untreated cerebral and cremaster VSMCs. Qualitatively both a decrease in β1 BKCa subunit activity and the cremaster muscle VSMC phenotype caused a rightward shift in the NPo vs. [Ca2+] relationship.
Analyses of open and closed times for cerebral artery VSMCs were performed on data sets obtained at +30 mV holding potential and values of free [Ca2+] ranging from 100 nm to 30 μm. As shown in Fig. 9, β-subunit-directed siRNA caused a decrease in channel open time (Fig. 9A) and a prolongation of shut time (Fig. 9B) compared with cerebral VSMCs treated with the control siRNA. β1-Subunit knockdown in cerebral VSMCs thus pushed these kinetic parameters towards values similar to those observed in native cremaster VSMCs.
The results of these studies demonstrate a significantly higher Ca2+ sensitivity of cerebral VSMC BKCa channels compared with that of cremaster VSMC. Thus, the cells from cremaster muscle exhibited a Ca2+ set point ([Ca2+] required for half-maximal channel opening at 0 mV) of 12.1 compared with 5.0 μm in cerebral VSMCs. Further differences in the biophysical properties in the two populations of vascular BKCa channels were a shorter open time and longer closed time in cremaster compared with cerebral VSMCs. β1-siRNA treatment of cerebral VSMCs to decrease β1-subunit protein expression led to a more ‘cremaster-like’ behaviour of BKCa activation, as shown by decreases in E2 sensitivity, STOC amplitude and frequency, and single-channel Ca2+ sensitivity. These data appear consistent with the previous demonstration of a higher ratio of β1-subunit:α-subunit of BKCa channels in cerebral compared with cremaster VSMCs (Yang et al. 2009). Functionally, at the channel level, the higher β1-subunit:α-subunit ratio results in greater Ca2+ sensitivity and a higher NPo of BKCa in the cerebral vasculature relative to that of skeletal muscle, providing an enhanced hyperpolarizing and vasodilating influence in cerebral vessels compared with those of skeletal muscle (Jackson & Blair, 1998; Yang et al. 2009). Greater BKCa channel activity in cerebral arteries would thus help maintain adequate blood flow in this vascular bed, and counteract the influence of vasoconstrictor mechanisms.
In the first series of studies, single BKCa channel recordings were obtained for both cremaster and cerebral VSMCs. Unitary BKCa channel amplitude was determined from amplitude histograms fitted with a Gaussian function. The conductance (γ) of BKCa channel was found to be similar (approximately 240 ps) in both vascular beds, and is consistent with values reported for BKCa in a number of previously published studies (Nelson & Quayle, 1995; Jackson & Blair, 1998; Wei et al. 2005). The observations of similar levels of single-channel conductance for BKCa, along with voltage sensitivity (Table 1), in the two VSMC preparations suggest that channel exhibits the same basic properties in these cell types. Differences in the electrophysiological characteristics were therefore assumed to lie at the level of channel regulation.
Subsequent studies examined the relationships between NPo and Vm at varying levels of [Ca2+]. These experiments showed BKCa channel open probability in cerebral VSMCs to be more sensitive to [Ca2+]i than that of cremaster VSMCs. Thus, at a given [Ca2+] concentration, the NPo vs. Vm curves were significantly left-shifted for cerebral VSMCs compared with that of cremaster VSMCs. In contrast to the differences in Ca2+ sensitivity, calculated Boltzmann constants (Z) were similar at each [Ca2+] in the two cell types, indicating that voltage dependence of the channel was not different between the two tissues.
HEK cell expression systems have been used previously to define the biophysical characteristics of BKCa comprised of only α-subunits compared with channels containing both α- and β-subunits. Nimigean and Magleby showed that co-expression of the α and β BKCa subunits caused a marked increase in mean single-channel open time (holding potential +30 mV) relative to expression of the α-subunit alone (Nimigean & Magleby, 1999). This effect was evident at all values of cytoplasmic [Ca2+] studied. Although an apparently more complex relationship was observed between mean closed time and [Ca2+], channels consisting of both the α and β BKCa subunits showed a decreased closed time at all values of [Ca2+] compared with channels containing the α-subunit alone. By comparing the data from the study of Nimigean & Magleby (1999; see Fig. 2) with the results of the present study (Fig. 3A and B), it is evident that the open and closed times of cerebral VSMC BKCa channels more closely resembles those of the α+β-subunit expression system, while the kinetics of the cremaster VSMC BKCa are more α-like in character. Thus, co-expression of the β1-subunit and α-subunits showed an approximate fourfold increase in channel open time ([Ca2+]= 5 μm; holding potential +30 mV) in the Nimigean and Magleby study, while in the present study open time under the same conditions was five times greater in cerebral VSMCs compared with that of cremaster VSMCs.
As an additional approach to characterize the relative behaviour of BKCa in cerebral and cremaster VSMCs, subunit-specific siRNA approaches were developed to manipulate expression of either the α- or β-subunit protein. The efficacy of the approach was initially verified by uptake of fluorescently labelled siRNA into SMCs and by demonstration of decreased protein expression by Western blotting. Further, to demonstrate functional effects at the vessel level, α-siRNA-treated vessels were shown to have enhanced myogenic tone relative to control arteries. Following this, whole-cell patch-clamp was used to show that decreasing expression of the α-subunit led to a decrease in macroscopic K+ current, decreased responsiveness to the BKCa channel agonist, NS-1619, and a decrease in STOC activity (assessed in terms of both frequency and amplitude). While a similar pattern was generally seen in both cell types, a relatively greater magnitude of effect was seen in the cerebral VSMCs compared with those of cremaster. This may relate to a greater baseline level of α-subunit protein expression in the cerebral VSMC, as reported in an earlier study (Yang et al. 2009). In response to the β-subunit-directed siRNA, only a slight reduction in cerebral basal K+ current was observed; however, a marked reduction in the stimulatory effect of oestrogen was apparent. Consistent with this observation, oestrogen has been previously shown to stimulate BKCa via an interaction with the β1-subunit (Valverde et al. 1999; Dick & Sanders, 2001; De Wet et al. 2006). Decreased expression of the β1-subunit had, in contrast, little effect on the stimulatory effects of NS-1619, which has been reported to act via the α-subunit (Gribkoff et al. 1996). Decreasing β1-subunit expression markedly decreased STOC frequency in cerebral VSMCs to a level similar to that of cremaster VSMCs. Thus, these studies at the whole-cell level suggest that the electrophysiological characteristics of the BKCa channel of cerebral VSMCs can be manipulated to resemble those of the cremaster cells by acute suppression of β1-subunit protein expression.
Interestingly, the β1-subunit BKCa-directed siRNA also significantly decreased the amplitude of the remaining STOCs in cerebral compared with cremaster muscle VSMCs. Thus, the siRNA treatment decreased STOC amplitude by approximately 68% in the cerebral VSMCs, but only 14% in the cremaster VSMCs. In absolute terms, after β-subunit siRNA treatment, STOC amplitude was not significantly different between cremaster and cerebral VSMCs (15.2 ± 2.2 and 18.6 ± 2.1 pA, respectively). Although not directly assessed in the present study, we showed previously (Yang et al. 2009) that cremaster VSMCs showed low-amplitude STOCs (relative to cerebral VSMCs) and did not exhibit measurable Ca2+ spark activity. In cerebral VSMCs, Perez et al. (1999) showed that while there was tight coupling between a Ca2+ spark and a subsequent STOC, they also observed a population of low-amplitude STOCs that was not associated with the transient Ca2+ events. While this situation may relate, in part, to technical issues such as Ca2+ sparks being imaged from a restricted optical plane while STOCs were recorded from a more three-dimensional environment, it is also conceivable that differences in BKCa channel populations rendered one population more susceptible to the effects of β1-subunit siRNA treatment.
To further confirm that suppression of β1-subunit expression leads to a decrease in Ca2+ sensitivity of cerebral VSMCs, additional studies were performed at the single-channel level. To ensure selection of cells containing the siRNA, these studies were performed using a fluorescently tagged siRNA directed at the β1-subunit of BKCa. These data showed that the siRNA treatment of cerebral VSMCs caused a rightward shift in the open probability vs. [Ca2+] relationship, resembling the pattern observed in freshly isolated cremaster VSMCs. A caveat to this analysis is that the siRNA does not lead to a total loss of the β1-subunit, and therefore comparisons are limited to a qualitative nature. However, these data were supported by the observed changes in open and closed times as well as the effects of siRNA treatment on the characteristics of STOCs.
From a methodological point of view, the current studies using siRNA-mediated protein knockdown focused on the effects of an acute reduction in BKCa channel subunit expression. This approach was taken to determine if channel activity could be manipulated in the absence of chronic alterations in channel function, such as in the genetically β1-subunit-deficient mouse (β1BKCa−/−; Brenner et al. 2000; Pluger et al. 2000), where adaptive/compensatory changes may occur over time. That being said, it is apparent that the BKCa channel in cremaster VSMCs exhibits similar functional characteristics to BKCa channels in cerebral VSMCs of β1BKCa−/− mice. Additional methodological considerations relate to the actual stoichiometry of subunit expression (i.e. number of β1- relative to α-subunits within a holo-channel complex) as the current approaches do not enable heterogeneity to be examined at this level. Although differing subunit stoichiometries may exist, a full complement of four β1-subunits per holo-channel is predicted to be required for maximal effect (Wang et al. 2002). Importantly, although Western blot analysis was necessarily conducted on homogenates of whole-vessel segments, the use of fluorescently-labelled β-subunit-directed siRNA allowed us to identify cells exposed to the reagent for use in single-channel analyses. Further, α-subunit-directed siRNA treatment enhanced myogenic tone in cannulated and pressurized artery segments (Fig. 4D), consistent with a physiological role of the channel in modulating vascular resistance.
In summary, the data are consistent with a higher ratio of β1-subunit:α-subunit of BKCa channels in cerebral compared with cremaster VSMCs. Functionally, this results in a greater Ca2+ sensitivity and a higher NPo of BKCa in the cerebral vasculature relative to that of skeletal muscle. Further, the studies highlight regional differences in vascular ion channel function that may contribute to differences in the respective mechanisms underlying local blood flow control (Kotecha & Hill, 2005). A detailed understanding of these differences may ultimately provide insight for the development of targeted therapies for the treatment of vascular pathologies.
M.A.H. (HL92241-03) and M.J.D. (P01 HL-095486) are supported by grants from the National Institutes of Health (NHLBI), and A.P.B. by grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council.