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Ca²⁺-activated K⁺ channel-associated phosphatase and kinase activities during development

¹Center for Perinatal Biology, ²Department of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, California

Submitted 22 October 2004 ; accepted in final form 8 February 2005

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
In ovine basilar arterial smooth muscle cells (SMCs), the fetal "big" Ca²⁺-activated K⁺ (BK) channel activity is significantly greater and has a lower Ca²⁺ setpoint than BK channels from adult cells. In the present study, we tested the hypothesis that these differences result from developmentally regulated phosphorylation of these channels. Using the patch-clamp technique and a novel in situ enzymological approach, we measured the rates and extents of changes in BK channel voltage activation from SMC inside-out patch preparations in response to selective activation and inhibition of channel-associated protein phosphatases and kinases (CAPAKs). We show that BK channel activity is modulated during development by differential phosphorylation and that the activities of CAPAKs change substantially during development. In particular, excised membrane patches from adult SMCs exhibited greater protein kinase A activity than those from a fetus. In contrast, fetal SMCs exhibited greater protein kinase G activity and phosphatase activity than adult SMCs. These findings extend our previous observation that the BK channel Ca²⁺ setpoint differs significantly in adult and fetal cerebrovascular myocytes and suggest a biochemical mechanism for this difference. In addition, these findings suggest that the functional stoichiometry of CAPAKs varies significantly during development and that such variation may be a hitherto unrecognized mechanism of ion channel regulation.

inside-out patch; protein kinase; smooth muscle cells

Previously, we reported that in ovine middle cerebral arteries, fetal myocytes depend more on the influx of extracellular Ca²⁺ during contraction than do myocytes of adults, whereas adult cells rely more upon Ca²⁺ mobilization from intracellular stores (1, 2, 24, 25). In particular, the inositol 1,4,5-trisphosphate-releasable and ryanodine-sensitive Ca²⁺ stores appear to be developmentally modulated (24, 27). Moreover, membrane potential and [Ca²⁺]_i are closely related such that depolarization is associated with increased [Ca²⁺]_i. Because of a relatively high membrane resistance, slight changes in K⁺ channel activity dramatically affect cell membrane potential (29). In particular, the "big" Ca²⁺-activated K⁺ (K_Ca or BK) channels respond to increases in [Ca²⁺]_i and hyperpolarize the membrane to inhibit additional Ca²⁺ entry, thereby coupling the control of membrane potential to changes in [Ca²⁺]_i. Thus the regulation of BK channel activity may be an important determinant of developmental differences in SMC activity.

Basilar arterial SMCs from neonatal rats exhibit a lower frequency of Ca²⁺ sparks, which activate the BK channels, compared with adult SMCs (18). Nonetheless, neonatal myocytes exhibit minute intracellular Ca²⁺ release events mediated by ryanodine receptors (18, 32). Adult SMCs appear to possess different releasable intracellular Ca²⁺ pools from those of the fetus (24, 27). Recently, we reported (21) that BK channels in fetal myocytes respond to lower [Ca²⁺]_i. The cause for this greater apparent sensitivity is that more of the BK channels in fetal SMCs are activated at a given [Ca²⁺]_i; that is, the BK channel Ca²⁺ setpoint is lower in fetal than in adult myocytes.

In the present study, we attempted to identify the underlying mechanism mediating changes in BK channel activity during development. Several studies have indicated that differential regulation of protein phosphatases (PPs) and kinases, including those that are channel associated, modulate BK channel activity (29). These possibilities lead us to hypothesize that in cerebral arteries, BK channel activity is modulated by phosphatases and cyclic nucleotide-dependent protein kinases in an age-dependent manner.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Experimental animals. For these studies, we used basilar arteries from near-term fetal (140 days of age) and nonpregnant adult female (<2 yr of age) sheep obtained from Nebeker Ranch (Lancaster, CA). All surgical and experimental procedures were performed within the regulations of the Animal Welfare Act and the National Institutes of Health's Guide for the Care and Use of Laboratory Animals, "The Guiding Principles in the Care and Use of Animals" approved by the Council of the American Physiological Society, and the Animal Care and Use Committee of Loma Linda University.

Isolating vascular SMCs. We enzymatically dissociated SMCs from dissected basilar arteries. This cell isolation procedure has been described previously (21). Briefly, basilar arteries from adults (200–400 µm diameter) and fetuses (150–200 µm diameter) were cut into 1-mm² pieces and placed in HEPES-buffered solution that contained 0.3 mg/ml papain and 1 mg/ml dithioerythritol for 20 min at 37°C. The tissues were then transferred to a second incubation in HEPES-buffered solution that contained 100 µM CaCl₂, 0.6 mg/ml collagenase type F, and 0.3 mg/ml collagenase type H (Sigma Blend Collagenase; Sigma; St. Louis, MO) and incubated for 15 min at 37°C. All of the enzymatic solutions also contained 1 mg/ml bovine serum albumin to minimize proteolytic effects on the cells. The digested tissues were triturated with a fire-polished, siliconized (Sigmacote; Sigma) glass Pasteur pipette to yield intact, separated SMCs. The cells were kept on ice and used within 6 h.

Single-channel recordings. We mounted coverslips that contained adherent SMCs for 15 min (in a perfusion chamber that contained HEPES-buffered solution) on the stage of an inverted microscope (Axiovert 35M; Carl Zeiss Instruments; Chester, VA), where they were viewed and verified to have characteristic elongated shapes with axial ratios of 10:1 for adult and 5:1 for fetal myocytes. The HEPES-buffered solution was then exchanged for the bathing solution.

We recorded single-channel currents in inside-out excised configuration from SMC membrane patches using an Axopatch 200B amplifier (Axon Instruments; Foster City, CA). Single-channel currents were recorded with Clampex 8 (Axon Instruments). Patch pipettes were fabricated from borosilicate glass capillary (10 cm length, 1.5 mm OD, and 0.86 mm ID; Sutter Instrument; Novato, CA) by using a programmable Flaming-Brown pipette puller and standard fire-polishing procedures. The pipette resistance for microscopic recording approximated 20 M. Currents were filtered at 5 kHz using an Axopatch 200B internal four-pole, low-pass Bessel filter and were digitized at 25 kHz. An agar salt bridge was used to minimize the solution junction-potential differences.

As we have reported (21), the number of channels present in any given excised patch (N) was estimated from all-points histograms. Channel activity (NP_o) was calculated from

where i is the number of open channels (0 for closed state), and A_i is the area associated with the curve-fit individual peak for each channel state. The single-channel open probability (P_o) was then calculated from NP_o/N. The values for N were obtained by using high depolarization potential (e.g., +100 mV) to ascertain that less than three coincidental open events occurred during long recordings (>20 s) at a P_o > 0.8. Preparations with more than three channels present were discarded.

Data were then expressed as channel activities (P_o) relative to maximum channel activity (P), where P is defined as the maximum P_o of a BK channel throughout a set of recording command potentials. Operationally, P is the P_o obtained by using a high depolarization potential (i.e., +100 mV). Voltage-activation curves of P_o/P vs. voltage were fitted to a form of the Boltzmann equation as P_o/P= {1 + exp[(V_1/2 – V_m)/K]}^–1, where V_1/2 is the membrane potential (V_m) required for half-maximal activation of the channels, and K is the logarithmic voltage sensitivity (i.e., V required for an e-fold increase in activity).

Macroscopic recording. We recorded macroscopic currents in inside-out configuration as for a single-channel recording, except that the pipette tip opening was larger. Patch pipettes were fabricated from borosilicate glass capillary tubing (10 cm length, 1.5 mm OD, and 1.17 mm ID) as described (see Single-channel recordings). The pipette resistance for the macroscopic recording approximated 2–3 M. Data from recorded currents were filtered at 1 kHz and acquired at 5 kHz. Conductance (G) values were calculated as G = I/V_m, where I is current. Dividing G values by G_max, where G_max is defined as the largest G value obtained in each experiment, normalized to each experiment. BK channel activity was expressed as relative conductance G/G_max. Voltage-conductance curves of G/G_max vs. voltage were fitted with a form of the Boltzmann equation G/G_max = {1 + exp[(V_1/2 – V_m)/K]}^–1.

The V_1/2 values were absolute values of the difference between the V_1/2 values at some time and at 0 min. The values of V_1/2 were plotted against the time of recording to yield the time course of change in voltage activation. The time course of V_1/2 values was best fit with a substituted form of the Michaelis-Menten equation, V_1/2 = V_max x t/(t_0.5 + t), where V_max is the maximum plateau of V_1/2, t_0.5 is the time it takes for the amplitude to rise to half of maximum amplitude, and t is the recording time in minutes.

Chemical reagents and solutions. Papain was obtained from Worthington Biochemical (Lakewood, NJ). Ca²⁺ standard kits and fura 2 were obtained from Molecular Probes (Eugene, OR). Purified PKG, KT-5720, KT-5823, okadaic acid (OA), and cypermethrin were obtained from Calbiochem (EMD Biosciences; San Diego, CA). Both purified catalytic subunit of PKA (PKAc) and PKA peptide inhibitor PKI were gifts from William H. Fletcher (13, 36). All other chemicals were obtained from Sigma-Aldrich. Purified PKG was preactivated in 0.05 mM cGMP-containing bathing solution before use. For cell isolation, the HEPES-buffered solution contained (in mM) 55 NaCl, 80 sodium glutamate, 5.6 KCl, 10 HEPES, 2 MgCl₂, and 10 glucose, pH 7.3 with NaOH. Both the pipette and bathing solutions contained (in mM) 140 KCl, 10 HEPES (pH 7.2), 1 Mg²⁺, and 5 EGTA, and a free Ca²⁺ concentration of 3 µM, which was estimated with Max Chelator Sliders software (C. Patton; Stanford University; Ref. 30) and confirmed fluorometrically using fura 2 and Ca²⁺ standard kits.

Data analysis and statistics. All presented data were calculated as means ± SE. In all cases, n refers to the number of replicate samples. Statistical comparisons were performed at the 95% confidence level using two-sample, unpaired Student's t-tests or ANOVA. Where ANOVA showed significance, Duncan's test was also performed to test for the significance among treatment and age groups. We verified that all sample populations were distributed normally. For comparisons of values that were not significantly different, power analyses were performed to confirm that statistical power was 0.7 and the probability of type II errors was acceptably small. Curve fitting was performed with GraphPad Prism 3 software (GraphPad Software; San Diego, CA).

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Voltage-activation curves of adult and fetal myocytes. To determine the activity of BK channels from adult and fetal SMCs, we used the microscopic inside-out, excised-patch, voltage-clamp technique. The bathing solution, which represented the cytosol, contained 3 µM free Ca²⁺. Figure 1A shows a representative recording of BK channel activity from a patch preparation derived from a fetal SMC. The BK channel activity from a patch immediately after it was excised, i.e., at 0 min (Fig. 1A, top), is compared with the channel activity from the same patch 20 min later (Fig. 1A, bottom). We expressed BK channel activity as P_o/P, plotted against membrane potential (Fig. 1B), and fitted the data to the Boltzmann equation as described (see METHODS). The slopes of the voltage-activation curves, which represent the voltage sensitivity of the channel, did not change with time or between adult and fetal SMCs. Table 1 presents the V_1/2 values for the activation curves of BK channels in adult and fetal SMCs. From the table, it can be seen that the channels from fetal myocytes shift to the right toward more-positive potentials (i.e., channel rundown) by approximately twice as much as those from the adult cells.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Time-dependent changes in "big" Ca2+-activated K+ (BK) channel activity. All recordings were made in symmetrical 140 mM KCl with the bath containing 3 µM Ca2+. A: representative voltage-clamp recordings of BK channel activity from an inside-out micropatch excised from fetal basilar arterial smooth muscle cells (SMCs). One BK channel was present in the micropatch preparation. Potential was held at +30 mV. Activity of the freshly excised BK channel (0 min; top) and the channel activity 20 min later from the same preparation (bottom) are shown. Dotted lines indicate the closed (C) and open (O) states of the channels. B: voltage-activation curves of inside-out micropatch preparations. Channel openings were recorded at different membrane potentials and are expressed relative to maximum channel activity [open probability (Po)/maximum Po (Pomax)]. Data are from BK channels of adult basilar artery SMCs () and fetal myocytes (). Lines connecting data points were fitted to a form of the Boltzmann equation (see METHODS); freshly excised BK channels (0-min controls; solid lines) and BK channels at 20 min postexcision (dashed lines) are shown. Data are means ± SE (vertical bars); n = 9 adult and 15 fetal samples.

View this table: [in this window] [in a new window]   Table 1. Summary of V values from adult and fetal myocytes

View larger version (28K): [in this window] [in a new window]   Fig. 2. Time course of shift in BK channel activity. All recordings were made in symmetrical 140 mM KCl with the bath containing 3 µM Ca2+. A: representative inside-out macroscopic recordings of BK channel activity from excised fetal SMCs at 0 and 30 min after patch excision. Macroscopic currents were elicited by a series of 10-mV depolarizing steps (–100 to +100 mV) from a holding potential of 0 mV. Recording protocol of –100 to +100 mV with 500-ms pulses every 5 s is shown (inset). B: representative BK channel voltage-conductance curves recorded at different times show a progressive rightward shift. Data were recorded for a series of membrane potentials at 0, 2, 4, 8, 15, and 30 min after excision of inside-out macroscopic patches from fetal SMCs. Relative channel activity was expressed as conductance (G)/maximum conductance (Gmax). Voltage-conductance curves were fitted with the Boltzmann equation to determine the values for the membrane potential (Vm) required for half-maximal channel activation (V1/2) and draw curves. C: time course of right shift of V1/2 values from BK channels of fetal SMCs. The V1/2 values were calculated by subtracting V1/2 values of the time of recording from V1/2 values of the 0-min control. Best-fit curve to a substituted form of the Michaelis-Menten equation in which vi was the initial slope of the fitted curve (solid line), Vmax was the maximum extent to which the voltage-conductance shifted, and t0.5 was the time required to attain half-Vmax (see METHODS).

The time course of the shift toward more positive potentials (i.e., rightward shift) was plotted as a rectangular hyperbola with the two asymptotes parallel to the x- and y-axes (Fig. 2C). The data were best fit with a substituted form of the Michaelis-Menten equation as described (see METHODS). The time course exhibited an initial period of relatively rapid change followed by an asymptotic approach to a maximum value of V_1/2 (V_max). If the spontaneous shift in the activation of the channels resulted from endogenous, channel-associated PP activity, then the initial rate (v_i) of change of V_1/2 for the BK channels from myocytes is the best measure of such channel-associated activity, because the steady-state assumption for available substrate is most likely to apply under initial reaction conditions.

To determine whether PP activity caused the shift of the BK channel voltage-activation curve, we tested the effects of PP inhibitors. In both adult (Figs. 3A and 4A) and fetal (Figs. 3B and 4B) SMCs, the rates and the extents of the rightward shifts of the voltage-activation curves were almost completely inhibited by 2 nM OA, which at this concentration is a relatively selective inhibitor of PP2A (12). Subtracting the OA-insensitive activity from the activity of the untreated controls, we plotted the OA-sensitive activity, which we assumed was due to endogenous PP2A activity (Fig. 4C). In the adult preparations, the OA-sensitive activity constituted 64% of the untreated rightward-shift activity, whereas for the fetal preparations, the OA-sensitive activity was 87%. As shown in Table 2, the initial rate for the OA-sensitive rightward shift in the fetal SMCs was 3.8 times greater than in those of adults. Increasing the concentration of OA to 100 nM, which inhibits both PP2A and PP1 activity (12), did not further affect these voltage-activation curves. The kinetic parameters of the residual OA-insensitive rightward-shift activity of both adult and fetus were similar and were decreased partially to the same extent by 0.5 nM cypermethrin, which is an inhibitor of PP2B activity.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Representative inside-out macroscopic recordings illustrate the time-dependent effects of okadaic acid (OA) on BK channels from adults (A) and fetuses (B). Macroscopic currents were elicited by a series of 10-mV depolarizing steps (–100 to +100 mV) from a holding potential of 0 mV using the same recording protocol as in Fig. 2A. Recorded currents are shown from two time points, 0 and 20 min, without (–OA) and with (+OA) OA present in the bathing solution. Vertical scale bars represent 500 pA for each set of recordings.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Time courses of BK channel rightward shifting from adult and fetal myocytes. As described for Fig. 2, voltage-conductance curves were recorded at different times from inside-out macropatches and then plotted as time-course curves. Membrane patches were pulled either in the presence of 2 nM OA to inhibit phosphatase activity or in the absence of OA (control) for both adults (A; n = 6) and fetuses (B; n = 7). Values from preparations treated with OA were subtracted from control values to yield OA-sensitive curves (C). Best-fit curves (solid lines) were calculated from a substituted form of the Michaelis-Menten equation (see METHODS).

View larger version (20K): [in this window] [in a new window]   Fig. 5. Effects of endogenous and exogenous PKA activity on BK channel activity. In each case, micropatch preparations from adult and fetal sheep were pretreated with alkaline phosphatase (350 U/ml; dashed lines) for 10 min to maximally dephosphorylate the channels. A: effects of endogenously activated, channel-associated PKA on channel activity. After phosphatase pretreatment, current-voltage relationships were recorded, then OA (0.1 µM), KT-5823 (1 µM), and ATP (0.5 mM) were perfused through the bathing medium for 10 min before current-voltage relationships were again recorded (n = 7 adult and 5 fetal samples). B: effects of fully activated channel-associated PKA on channel activity was studied with addition of 0.5 mM cAMP. All treatments were the same as in A except that 0.5 mM cAMP was incubated with the OA, KT-5823, and ATP (n = 5 adults and 5 fetuses). C: effects of exogenous PKA catalytic subunit (PKAc) on channel activity. All treatments were the same as in A except that PKAc (30 U/ml) was incubated with the OA, KT-5823, and ATP (n = 6 adult and 10 fetal samples). Best-fit curves to the Boltzmann equation are shown (solid lines).

To determine whether PKA is associated with BK channels in the patch preparations, we added ATP (0.5 mM) to the perfusing solution in the presence of the PKG inhibitor KT-5823 (1 µM) and OA (0.1 µM) after dephosphorylation with alkaline phosphatase. Figure 5A shows the dephosphorylated and subsequent leftward-shifted KT-5823 plus ATP-treated voltage-activation curves for both adult and fetal SMCs. The leftward shift of the KT-5823 plus ATP-treated channels was significantly greater for channels from adult than fetal SMCs (Fig. 5A and Table 1). Such a leftward shift suggests phosphorylation by endogenously activated PKA. We also attempted to differentiate between endogenously activated PKA and total available PKA activities by adding 0.5 mM cAMP to fully activate all available PKA (Fig. 5B). The slightly but not significantly greater leftward shift of both adults and fetuses in the presence of added cAMP suggests that for all practical purposes, the channel-associated PKA activity was either endogenously activated or in the form of the catalytic subunit.

To determine whether the channels were maximally phosphorylated by the fully activated endogenous PKA, we exposed phosphatase-treated patch preparations to PKAc (30 U/ml) in the presence of ATP, OA, and KT-5823. Figure 5C shows that added PKAc leftward shifted the BK voltage-activation curves from both adult and fetal SMCs to a similar extent (see Table 1). The added PKAc leftward shifted the adult BK channels to the same extent as that of fully activated endogenous PKA (see Table 1), which suggests that fully activated endogenous PKA has access to all of the PKA phosphorylation sites. However, that PKAc shifted the fetal channel activation to a greater extent than that for fully activated endogenous PKA (Fig. 5B) suggests that something prevented fetal BK channels from being as fully phosphorylated by channel-associated PKA. In experiments designed to measure the effects of endogenously activated kinases on channel activity, we did not attempt to inhibit endogenous phosphodiesterase activity, because we had found that 3-isobutyl-1-methylxanthine (IBMX, 300 µM) had no significant effect on the extent of shifting of the voltage-activation curves 20 min after patch excision (V_1/2: control, 20.6 ± 6.5; IBMX, 20.6 ± 1.0 mV; n = 3). In addition, in experiments designed to fully activate protein kinases, the levels of added cyclic nucleotides were high enough to override endogenous phosphodiesterase activity in the patch (6).

Channel-associated PKG activity. We also assayed the effects of endogenous channel-associated PKG activity on BK channels in a manner similar to that for channel-associated PKA activity. After dephosphorylation with alkaline phosphatase, we determined the BK voltage-activation curves in the presence of the selective PKA inhibitor KT-5720 (0.3 µM), OA (0.1 µM), and ATP (0.5 mM). Figure 6A shows that treatment with KT-5720 plus ATP leftward shifted the voltage-activation curves for both adult and fetus from the alkaline-phosphatase-dephosphorylated state. The almost twofold greater leftward shift of the fetal BK channel voltage-activation curves suggests that these BK channels are associated with a higher level of endogenously activated PKG activity than are channels from adults.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Effects of endogenous and exogenous PKG activity on BK channel activity. In each case, micropatch preparations from adult and fetal sheep were pretreated with alkaline phosphatase (350 U/ml; dashed lines) for 10 min to maximally dephosphorylate the channels. A: effects of endogenously activated channel-associated PKG on channel activity. After phosphatase pretreatment, current-voltage relationships were recorded, then OA (0.1 µM), KT-5720 (0.3 µM), and ATP (0.5 mM) were perfused through the bathing medium for 10 min before current-voltage relationships were again recorded (n = 4 adult and 4 fetal samples). B: effects of fully activated channel-associated PKG on channel activity was studied with addition of 0.05 mM cGMP. All treatments were the same as in A except that 0.05 mM cGMP was incubated with the OA, KT-5720, and ATP (n = 5 adult and 4 fetal samples). C: effects of exogenous PKG on channel activity. All treatments were the same as in A except that PKG (2,000 U/ml) was incubated with the OA, KT-5720, and ATP (n = 8 adult and 12 fetal samples). Best-fit curves to the Boltzmann equation are shown (solid lines).

Time courses of protein kinases on BK channels. In a manner similar to our study of endogenous PP activity (see Figs. 2, 3, and 4), we used the macroscopic inside-out patch technique to study the time course of associated kinase-mediated effects on BK channel activity. After initial BK channel dephosphorylation with alkaline phosphatase, we added ATP (0.5 mM) in the presence of OA (0.1 µM) and recorded the voltage-conductance curves at specified times. The resulting leftward shift in terms of V_1/2 values (V_1/2) for channels from adult and fetal SMCs is shown in Fig. 7A as a time course. The initial slopes of the rising phases of the time course reflect the initial rate (v_i) of change of V_1/2 values. The initial rate for BK channels from adult cells was three times that from fetal cells (Table 3), although the maximum extent to which the channels were leftward shifted was similar. Repeating the experiment in the presence of KT-5823 and KT-5720 plus ATP and OA blocked a detectable leftward shift in both adult (V_1/2 = 4.5 ± 6.3 mV; n = 4) and fetal (V_1/2 = 2.9 ± 7.3 mV; n = 4) SMCs. This strongly suggests that the leftward-shift activity of ATP was due to the actions of partially activated PKA and/or PKG.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Time courses of endogenous kinase-mediated BK channel leftward shifting. Voltage-conductance curves were recorded at different times from inside-out macropatches after pretreatment with alkaline phosphatase as described for Fig. 5, and the V1/2 values were calculated from the Boltzmann equation as described for Fig. 2B and then plotted as time-course curves as described in Fig. 2C. A: effects of endogenously activated total protein kinase activity on time course of BK channel leftward shifting. Channel activities were measured and displayed in the presence of OA (0.1 µM) and ATP (0.5 mM); n = 5 adult and 5 fetal samples. B: effects of endogenously activated PKA activity on time course of BK channel leftward shifting. Channel activities were measured and displayed as in A except that KT-5823 (1 µM) was also included; n = 5 adult and 5 fetal samples. C: effects of endogenously activated PKG activity on time course of BK channel leftward shifting. Channel activities were measured and displayed as in B except that KT-5720 (0.3 µM) was also included; n = 5 adult and 5 fetal samples. Lines were best fit to a substituted form of the Michaelis-Menten equation (as described in METHODS).

By performing the same experiment in the presence of ATP plus KT-5823 to inhibit endogenous PKG activity, we measured the time course of the effect of partially activated, endogenous PKA on channel activity (Fig. 7B). Likewise, in the presence of ATP plus KT-5720, to inhibit endogenous PKA activity, we measured the time course of the effect of partially activated, endogenous PKG (Fig. 7C). In the adult preparation, the initial rate of the leftward shift in the presence of KT-5823 was at least equal to that of ATP alone, as was its maximum extent (Fig. 7B; Table 3). The initial rate in the presence of KT-5720 was <10% that of ATP alone, whereas the maximum extent of the shift was only 40% that of ATP alone (Fig. 7C). Thus in adult SMCs, the activity of partially activated PKA was much greater than that for partially activated PKG. In fetal SMCs, we measured comparable levels of partially activated PKA (Fig. 7B) and PKG (Fig. 7C) activities. In addition, both the partially activated PKA and PKG leftward shifted channel activation to the same extent but only to 75% that of ATP alone (Table 3).

By adding cAMP and cGMP together, we measured the time course of the effect of the fully activated, channel-associated PKA and PKG on BK channel activity. In both adult and fetal preparations, the initial rates of fully activated PKA and PKG activities (Fig. 8A; Table 3) were about three times greater than those of the control endogenous activity (see Fig. 7A; Table 3) and were higher than those of partially activated PKA (see Fig. 7B) or PKG (see Fig. 7C) alone. Nonetheless, the initial rates of the fully activated PKA and PKG from adults were 2.5 times greater than those of fetuses.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Time courses of endogenous kinase-mediated BK channel leftward shifting. Voltage-conductance curves were recorded at different times from inside-out macropatches as described for Fig. 6. A: effects of fully activated endogenous PKA and PKG activity on time course of BK channel leftward shifting. Channel activities were measured and displayed in the presence of OA (0.1 µM), ATP (0.5 mM), cAMP (0.5 mM), and cGMP (0.05 mM); n = 5 adult and 5 fetal samples. B: effects of fully activated endogenous PKA activity on time course of BK channel leftward shifting. Channel activities were measured and displayed in the presence of OA (0.1 µM), ATP (0.5 mM), cAMP (0.5 mM), and KT-5823 (1 µM); n = 4 adult and 5 fetal samples. C: effects of fully activated endogenous PKG activity on time course of BK channel leftward shifting. Channel activities were measured and displayed in the presence of OA (0.1 µM), ATP (0.5 mM), cGMP (0.05 mM), and KT-5720 (0.3 µM); n = 5 adult and 5 fetal samples. Lines were best fit to a substituted form of the Michaelis-Menten equation (as described in METHODS).

In a similar manner, by using KT-5720 plus cGMP, we measured the time course of the effects of the fully activated, endogenous PKG on BK channel activity (Fig. 8C). In adults, the level of fully activated PKG activity was almost three times higher than that of partially activated PKG activity (Table 3). In addition, the level of partially activated PKG activity in adults was one-third that of fetuses, as was the level of the fully activated PKG. However, the maximum extents to which the fully activated channel-associated PKG activity leftward shifted the channel voltage-conductance curves were similar in both age groups (Table 3).

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
We have used a novel on-patch enzymological approach to measure the rates and extents to which the voltage-activation curves of BK channels change in response to selective activation of channel-associated PPs and kinases. We show for the first time that BK channel activity is modulated during development by differential phosphorylation and that the activities of channel-associated phosphatases and kinases (CAPAKs) change substantially. These findings augment our previous observation that the BK channel Ca²⁺ setpoint differs significantly in adult and fetal myocytes (21) and suggest a biochemical mechanism for this difference. More generally, these findings offer mechanistic insights into the physiological changes that accompany cerebrovascular SMC development.

Channel-associated phosphatases and kinases. Because our experiments employed cell-free, inside-out membrane-patch preparations, the effects of endogenous PPs and kinases most likely originated from the cytoplasmic side of excised membrane patches. PPs and kinases have been reported to be anchored in the plasma membrane (45) and bound directly to the COOH terminus of BK -subunits (44, 47) or via an anchoring protein (41). Because we do not know the exact means of PP and kinase attachment, we refer to them as CAPAKs.

Channel-associated PP activity. At the time of patch excision, BK channel P_o was significantly higher in SMCs from fetuses than those from adults (see Table 1 and Fig. 1). This is consistent with whole cell measurements of BK channel activity being higher in fetal than in adult basilar myocytes (21). After patch excision, BK channel activity decreased as is reflected in a progressive rightward shift of the channel voltage-activation curve (see Fig. 1) that is inhibited by low levels of OA (see Fig. 4). These changes in channel activity suggest that the channels are modified by channel-associated PP activity.

Channel-associated protein kinase activity. The presence of channel-associated PP activity implies the existence of an associated protein kinase activity. To study these kinases, we first attempted to totally dephosphorylate the BK channels by adding alkaline phosphatase. This rightward shifted channel activation to a greater extent than that caused by channel-associated PP activity (see Fig. 5A). Subsequent addition of ATP leftward shifted the channel-activation curves. The selective PKA and PKG inhibitors, KT-5720 and KT-5823, respectively, when added together, completely blocked the ability of ATP to leftward shift channel activation from either adults or fetuses (5, 37, 39); however, neither of these inhibitors alone could fully block leftward shifting in the presence of ATP. This suggests that both PKA and PKG activities are present in the patch preparations and are at least partially activated (3, 4).

To study specific channel-associated protein kinase activity and to eliminate possible cyclic nucleotide cross-activation (42), we inhibited PKG when we wanted to measure only PKA activity and vice versa. This approach enabled us to assess both the relative amount of channel-associated specific protein kinase activity and the extent to which each was endogenously activated.

Channel-associated PKA activity. The initial rate of the leftward shift by endogenously activated PKA was approximately four times higher in adults than in fetuses (see Table 3). With the addition of cAMP to activate fully channel-associated PKA activity, the rates of shifting increased 2.5-fold in adults and 3.3-fold in fetuses. This suggests that in both adults and fetuses, about one-third of the total channel-associated PKA was endogenously activated, and the total amount of channel-associated PKA in adults was about three times that of fetuses.

Channel-associated PKG activity. The initial rate of leftward shift by endogenously activated PKG was threefold greater in fetuses than in adults (see Table 3). The addition of cGMP to fully activate the channel-associated PKG activity increased the rate of leftward shift 2.8-fold in adults and 3.0-fold in fetuses. This suggests that in both age groups, about one-third of the total channel-associated PKG was endogenously activated. In addition, the total amount of channel-associated PKG in adults was about one-third that of fetuses. Together, these results imply that BK channels in adult SMCs depend more on activation by PKA, whereas those of the fetus depend more on PKG.

Kinase-specific vs. generalized phosphorylation sites. The BK channel phosphorylation sites have not been identified fully in part because of differing splice variants of the -subunit (42). The -subunit is expressed from one gene that includes at least five splice sites (10, 28, 34). In SMCs, the -subunit is associated with ₁-subunits (8, 9, 16, 20, 40), both of which possess phosphorylation sites (35, 43).

Splice variants can respond differently to PKA phosphorylation. For instance, in mouse anterior pituitary corticotrope AtT20 cells, BK channels are inhibited rather than activated by PKA (14, 38). In contrast, in Chinese hamster ovary cells, neither PKG nor PKA affect the activity of coexpressed human myometrial BK channel - or -subunits (46). Thus some splice variants may lack PKA phosphorylation sites, or the conformations of splice variants may allow only certain sites to be phosphorylated (42). In addition, phosphorylating one site may mask the effect of phosphorylating other sites (41).

Although BK channel splice variants are known to express different activities and phosphorylation sites, we do not believe that the differences between adult and fetal channels reported in the present study result from such variants. Channel activities from both age groups shifted to the same extent when treated with different exogenous protein kinases or with alkaline phosphatase. This indicates that the channels from the two age groups respond similarly to treatments that result in maximal phosphorylation or dephosphorylation. This also implies that the reported differences between the two age groups may result from factors other than the expression of different channel isoforms.

We reasoned that if, on the one hand, channel-associated PKA and PKG phosphorylate exclusively different sites on BK channels, then PKA and PKG together in the presence of ATP should produce both an initial rate (v_i) and a maximum leftward shift (V_max) that would approximate the sum of the v_i and V_max values of the respective endogenous kinases alone. On the other hand, if PKA and PKG compete for the same phosphorylation sites, then activating them together would produce v_i and V_max values that would approximate those of the more active of the two kinases acting alone. As indicated by the results in Table 3, the v_i and V_max values of the endogenously activated kinases together in both adults and fetuses are not significantly different from those of the PKA values in adults or, in fetuses, from the PKA or the PKG values alone. Assuming that no downstream factor limits the effect of channel phosphorylation, we hypothesize that the channel-associated PKA and PKG phosphorylate the same sites.

Extents of channel leftward shifting. The extent of the channel leftward shift may reflect the extent of channel phosphorylation. Channel-associated PKA, whether partially or fully activated, leftward shifts BK channels from adult preparations to a greater extent than corresponding fetal preparations (see Table 3). On the other hand, in fetuses, channel-associated PKG, whether partially or fully activated, leftward shifts BK channels to the same extent as fully activated PKG in adults, but significantly more than partially activated PKG in adults. Possible mechanisms that could explain the differences in extents of phosphorylation in the two age groups include 1) channels from adults and fetuses present different phosphorylation sites, 2) CAPAKs sterically block some of the channel phosphorylation sites, 3) different levels of associated kinase activity cause different extents of phosphorylation, and 4) channel-associated kinases have restricted access to phosphorylation sites.

Because adult and fetal channels are leftward shifted to the same extent by exogenous PKAc or PKG (see Table 1), it seems unlikely that the channels either present different phosphorylation sites or that the CAPAKs sterically block some of the channel phosphorylation sites. Also, it seems unlikely that the different extents of channel leftward shifting result from different levels of associated kinase activity, as low levels of PKA activity in fetuses or low levels of PKG activity in adults should be able to maximally leftward shift BK channels if provided enough time. On the other hand, associated kinases may have restricted access to channel phosphorylation sites due to the manner in which they associate with the channels. PKA can anchor or bind to the COOH terminus of the BK channel -subunit (41, 47), and more than one kind of protein kinase can bind directly to BK channels to form a signaling complex (22, 44). Such direct associations of kinases with the channel may restrict their access to some channel phosphorylation sites, whereas exogenous kinases would not have such constraints.

Perspectives. The present study demonstrates that 1) BK channels of ovine basilar artery SMCs are endogenously phosphorylated, 2) channel-associated PP2A activity is almost four times greater in fetal than in adult SMCs, 3) the total activity of BK channel-associated PKA in adults is three times greater than in fetuses, 4) the total activity of channel-associated PKG in fetuses is three times greater than in adults, 5) for both adults and fetuses, the endogenously activated levels of channel-associated PKA and PKG are about one-third that of the fully activated PKA and PKG activities, and 6) the activities of adult and fetal BK channels are altered to similar extents by purified, exogenous PKA and PKG and alkaline phosphatase.

Taken together, these findings indicate that the functional stoichiometry of the CAPAKs changes significantly during the course of development. This is represented diagrammatically in Fig. 9 for BK channels of the two age groups. It is likely, therefore, that BK channel activity is regulated during development not only by differential phosphorylation mediated by CAPAKs under the control of SMC signaling pathways but also by differential association with PPs and kinases. Thus it would appear that the dynamic state of channel phosphorylation is not only potentially influenced by the regulation of cellular signaling pathways but also by the selective and local association of PPs and kinases, which in the present study, we have shown changes during the transition from near-term fetus to adult.

View larger version (25K): [in this window] [in a new window]   Fig. 9. Diagram of channel-associated protein phosphatase and kinase differences between BK channels from adult and fetal vascular SMCs. For simplicity, only one BK -subunit of the BK tetrameric channel is illustrated. Diagram illustrates differences in the ratios of channel-associated protein phosphatase (), protein kinase A (), and PKG () between adults and fetuses. Functional ratios do not necessarily indicate molecular ratios, and the location of channel-associated proteins is not clear at present.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
National Institutes of Health Grant HD/HL-03807 (to L. D. Longo) supported this work.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Michael E. Barish (Beckman Research Institute; City of Hope, Duarte, CA) for helpful discussions and suggestions and Dr. William H. Fletcher for providing purified protein kinases. The authors also thank Brenda Kreutzer for assistance in preparation of the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. D. Longo, Center for Perinatal Biology, School of Medicine, Loma Linda Univ., Loma Linda, CA 92350 (E-mail: llongo{at}som.llu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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