Stimulation of unitary T-type Ca2+ channel currents by calmodulin-dependent protein kinase II

Paula Q. Barrett1, Hong-Kai Lu1, Roger Colbran2, Andrew Czernik3, and Joseph J. Pancrazio4

1 Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22903; 2 Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232; 3 Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021; and 4 Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, District of Columbia 20375


    ABSTRACT
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The effect of Ca2+/calmodulin-dependent protein kinase II (CaMKII) stimulation on unitary low voltage-activated (LVA) T-type Ca2+ channel currents in isolated bovine adrenal glomerulosa (AG) cells was measured using the patch-clamp technique. In cell-attached and inside-out patches, LVA channel activity was identified by voltage-dependent inactivation and a single-channel conductance of ~9 pS in 110 mM BaCl2 or CaCl2. In the cell-attached patch, elevation of bath Ca2+ from 150 nM to 1 µM raised intracellular Ca2+ in K+-depolarized (140 mM) cells and evoked an increase in the LVA Ca2+ channel probability of opening (NPo) by two- to sixfold. This augmentation was associated with an increase in the number of nonblank sweeps, a rise in the frequency of channel opening in nonblank sweeps, and a 30% reduction in first latency. No apparent changes in the single-channel open-time distribution, burst lengths, or openings/burst were apparent. Preincubation of AG cells with lipophilic or peptide inhibitors of CaMKII in the cell-attached or excised (inside-out) configurations prevented the rise in NPo elicited by elevated Ca2+ concentration. Furthermore, administration of a mutant recombinant CaMKIIalpha exhibiting cofactor-independent activity in the absence of elevated Ca2+ produced a threefold elevation in LVA channel NPo. These data indicate that CaMKII activity is both necessary and sufficient for LVA channel activation by Ca2+.

glomerulosa cells; low voltage-activated channels


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INTRODUCTION
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IN ADRENAL GLOMERULOSA (AG) cells, sustained Ca2+ entry through low voltage-activated (LVA) T-type Ca2+ channels has been hypothesized to regulate the synthesis and secretion of the steroid hormone aldosterone (5). Whole cell LVA currents recorded in AG cells at 37°C using physiological Ca2+ as the charge carrier (1.2 mM Ca2+) revealed voltage dependencies of activation (V1/2 = -50 mV) and inactivation (V1/2 = -68 mV) such that channel open probability (Po) was <0.3% at membrane potentials (Vm) negative to -79 mV (5). Since the Vm of AG cells established by physiological K+ solutions ranged from -98 to -79 mV (in HEPES-based Ringer solution), these data suggested little contribution of LVA channels to Ca2+ entry in the unstimulated steady state (5). Nevertheless, a small shift in the LVA channel half-activation potential could result in a substantial increase in Ca2+ entry, even in the absence of a change in Vm, since the foot of the voltage dependence of activation curve determines the extent of channel opening at negative membrane voltages. Therefore, an effective way to regulate LVA channel activity in AG cells during cell stimulation would be to effect a hormone-elicited shift in the half-activation potential. Indeed, in AG cells, activation of protein kinase C (39) reduces LVA channel activity while activation of Gi (26) or Ca2+/calmodulin-dependent protein kinase II (CaMKII) (27) enhances LVA channel activity by inducing a corresponding shift in the half-activation potential of LVA Ca2+ channels. Activation of CaMKII evokes a 10-mV hyperpolarizing shift in the half-activation potential (5, 27), resulting in an augmentation of the channel Po to 2.2% (5). Thus at a Vm of -74 mV, CaMKII-activated channels are eight times more likely to be open than under control conditions (5).

Angiotensin II (ANG II) is the major physiological regulator of aldosterone secretion. ANG II activates CaMKII (10), and CaMKII inhibition attenuates ANG II-stimulated aldosterone secretion (37). Therefore, CaMKII-mediated shifts in LVA channel gating would appear to mediate the control of Ca2+ entry underlying the physiological control of aldosterone secretion.

In this paper, to begin to delineate the signaling cascade responsible for the action of CaMKII on whole cell LVA currents, we evaluated the necessity for additional downstream Ca2+-dependent effectors in the signaling pathway by examining the effect of CaMKII on single-channel activity. We observed that CaMKII activation increased the frequency of channel opening (NPo) with no apparent change in modal states or open-state dwell times. Our data suggest that CaMKII activation is both necessary and sufficient for LVA channel activation by Ca2+ and that CaMKII activity may be tightly associated with the plasma membrane close to the LVA Ca2+ channel.


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Cell isolation. Calf AG cells (2-7 days old) were prepared by collagenase digestion as described previously (10). Thin layers of zona glomerulosa were prepared from adrenal cortex and collected in a Ca2+-free Krebs-Ringer bicarbonate (KRB) that contained (in mM) 120 NaCl, 25 NaHCO3, 3.5 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 1.25 CaCl2, 0.1% dextrose, and 0.2% bovine serum albumin (BSA), equilibrated with 95% air-5% CO2 (pH 7.4). The slices were digested for 10 min at 37°C in KRB that contained 0.625 mM CaCl2, 0.1% BSA, and 0.35 U/ml collagenase (Worthington, Freehold, NJ). The cells were dispersed mechanically, filtered through a 20-µm nylon mesh filter (Tetko, Elmsford, NY), collected by centrifugation, and resuspended in KRB that contained 1.25 mM CaCl2 and 0.2% BSA. The remaining slices were digested again (10 min at 37°C in KRB that contained 1.25 mM CaCl2, 0.1%BSA, and 0.35 U/ml collagenase), and the dispersion and filtration procedures were repeated. Cells recovered from the second digestion were collected and further purified on a two-step (30%/56%) discontinuous Percoll gradient (Pharmacia, Piscataway, NJ) and resuspended in standard KRB buffer. Cells were either cultured on glass coverslips in serum-free media (1:1 DMEM/F-12 that contained 200 µg/ml amikacin and 200 µg/ml ticarcillin) for 2 h before experiments or cultured overnight on glass coverslips in media (1:1 DMEM/F-12 that contained 10% horse serum, 2% fetal calf serum, 200 µg/ml amikacin, and 200 µg/ml ticarcillin) and serum deprived for 6 h before patch voltage-clamp studies were begun.

Recording. AG cells adherent to noncoated glass coverslips were transferred to a 300-µl volume RC-25 recording chamber (Warner Scientific, New Haven, CT) that was continuously perfused by gravity at a rate of 0.5 ml/min (5). Electrical recording was performed at room temperature. Patch electrodes were pulled from aluminosilicate glass (1724; Garner Glass, Claremont, CA) on a two-stage puller (L/M-3-PA; List-Medical, Darmstadt-Eberstadt, Germany) coated with Sylgard (Dow Corning, Midland, MI) and fire polished. The electrode resistances were 5-10 MOmega after heat polishing. Single-channel currents were recorded with an EPC-7 patch-clamp amplifier (List-Electronic, Darmstadt-Eberstadt, Germany) filtered with an eight-pole low-pass Bessel filter (Frequency Devices, Haverhill, MA) set at a cut-off frequency (-3 dB) of 1.0 kHz and digitized at intervals of 80-100 µs. Pulse generation and data acquisition were performed using a ZEOS 486 computer with pCLAMP 6.0 software and a Digidata interface (Axon Instruments, Foster City, CA). Depolarizing test pulses (-35 mV or -40 mV) of 150-ms duration were delivered every 4 s from the holding potential of -90 mV unless otherwise indicated.

Solutions. A high-K+ external solution was used to lower the Vm to 0 mV (13, 16). The external solution contained (in mM) 110 potassium aspartate, 20 potassium chloride, 10 HEPES, 10 EGTA, 5 dextrose, and 1 magnesium chloride (pH adjusted to 7.4 with KOH). The Ca2+ concentration in the external solution was adjusted to 50 nM, 150 nM, 500 nM, and 1 µM depending on the experiments. Free Ca2+ in the external solution was calculated using the ligand binding program, EQCAL (Biosoft, Ferguson, MO). In the excised configuration, 1 mM ATP, 200 µM GTP, and 0.2 µM CaM were added to the external solution unless otherwise specified. The pipette solution contained (in mM) either 110 BaCl2 and 10 HEPES or 110 CaCl2 and 10 HEPES (pH adjusted to 7.4 with CsOH).

Data analysis. Idealized current traces were constructed from digitized records using the Transit 2.0 suite of programs (44). Channel openings or closings were identified using a slope detector to localize transitions between levels and a relative amplitude criterion to remove spurious transitions (44). Capacitative and leakage currents were adequately eliminated from the records by ensemble averaging records free of channel openings and subtracting this average from all the records. Idealized records were concatenated into a single file, using the Convide program provided by Dr. A. M. VanDongen. The concatenated file was used for NPo and burst analysis by Transit. Open channel current amplitudes for current-voltage (I-V) relationships were measured by manually fitting cursors to well-resolved channel openings. The amplitudes of detected events were binned and fitted to one or the sum of two Gaussian distributions using SigmaPlot for Windows version 5 (SPSS, Richmond, CA) {f = A1/sqrt(2pi )sigma 1[e - (x-µ1/sigma 1)2/2] + A2 /sqrt(2pi )sigma 2[e - (x-µ2/sigma 2)2/2]}; where sigma 1 and sigma 2 = SD of Gaussian distribution 1 and 2 respectively; µ1 and µ2 = mean of Gaussian distribution 1 and 2 respectively; A1 and A2 = amplitude term 1 and 2. For 27 nM Ca2+ (µ = 0.51 ± 0.06 pA, sigma  = -0.15 ± 0.006, A = 10.51); for 1.2 µM Ca2+ (main conductance: µ = 0.47 ± 0.33 pA, sigma  = -0.173 ± 0.003, A = 38.16 ± 0.66; subconductance: µ = 0.20 ± 0.12 pA, sigma  = -0.021 ± 0.0018, A = 2.83 ± 0.27). Where appropriate, data are given as means ± SE, and statistical significance was determined using Student's t-test with P < 0.05 considered significant. A statistical comparison of the proportion of blank sweeps under control and treatment conditions was accomplished by testing the difference between proportions (45), with P values <0.05 considered significant.

Measurement of intracellular Ca2+ concentration. AG cells were plated on collagen-coated glass coverslips and loaded as previously reported (10) for 30 min at room temperature with 4 µM fura PE3-AM (Teflabs, Austin, TX), a Ca2+-sensitive fluorescent dye with limited cell leakage. Cells were incubated in KRB for 90 min at 37°C to allow for the intracellular generation of the Ca2+-sensitive form of the dye. Coverslips were mounted in a 500-µl recording chamber (Medical Systems, Greenvale, NY), and adherent AG cells were perfused by gravity with an external patch solution that fixed extracellular Ca2+ at 50 nM or 1 µM. Fluorescence emission at >= 400 nm was measured in response to alternate excitation at 340 or 380 nm using a Deltascan 4000 spectrofluorometer (Photon Technology International, South Brunswick, NJ) and digitized at a rate of 5 Hz. Single cell intracellular Ca2+ concentration ([Ca2+]i) 340/380 fluorescence ratios were converted to nanomolar [Ca2+]i values according to standard calibration protocols (43). All recordings were performed at room temperature.

Preparation of T286D-CaMKIIalpha . T286D-CaMKIIalpha , which introduces an acidic aspartate residue in place of Thr-286, was expressed in Sf9 insect cells using baculovirus and then purified as described for the wild-type kinase (2). The purified baculovirus-expressed enzyme expressed 8-25% Ca2+-independent activity toward exogenous substrates, thereby partially mimicking the effect of Thr-286 autophosphorylation. The purified kinase (specific activity of 3-6 µmol · min-1 · mg-1 in the presence of Ca2+/CaM) was frozen in small aliquots in liquid N2 and then stored at -80°C. Thawed aliquots were diluted 100-fold in external patch solution such that the final concentration of the T286D-CaMKIIalpha subunit was ~60 nM.


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Properties of the LVA Ca2+ channel in AG cells. LVA Ca2+ channels displayed diverse patterns of small openings in response to weak test depolarizations. Figure 1 shows current records selected to illustrate the different types of gating behavior observed in a cell-attached patch of a neonatal calf AG cell where only a single channel appears to be active. Figure 1A illustrates the effect of holding potential and 1C the effect of driving force on the elicited currents. With 110 mM BaCl2 in the pipette, 150-ms depolarizations to -40 mV from a holding potential of -90 mV evoked currents of small amplitude. Consistent with whole cell currents in AG cells that show slow activation and inactivation at potentials negative to -30 mV (26), channel opening exhibited a long first latency and slow voltage-dependent inactivation. In a few records, repeated events were observed. Many records had no channel activity, indicating a low probability of opening at -40 mV. In some cell-attached patches (15%), larger conductance openings (presumably L-type) were observed, but in no patches were these openings frequent or observed alone. The complete disappearance of small conductance openings at a holding potential of -40 mV during an interpulse interval of 6 s (Fig. 1A, right) and their full recovery upon repolarization to -90 mV (data not shown) indicated the presence of potential-dependent inactivation. As shown in Fig. 1B, the amplitude histogram of the single-channel events at -35 mV was well fitted to one Gaussian distribution with a mean value of -0.51 ± 0.061 pA. Replacing BaCl2 (closed circles) with CaCl2 (open circles) in the patch pipette solution yielded equivalent values for the amplitude of single-channel openings (Fig. 1C). Construction of a single-channel I-V curve from current amplitudes measured at various test potentials defined a slope conductance of 8.7 pS in 110 BaCl2 (Fig. 1C). This value compared favorably with conductance values reported previously for native LVA Ca2+ channels recorded in glomerulosa cells (8 pS, 8.3 pS) (29, 30), chick sensory neurons (8.5 pS) (13), ventricular myocytes (8 pS) (32), adult hippocampal neurons (8.7 pS) (11), and for the recently cloned alpha 1H T-type Ca2+ channel (9 pS) (46). Overall, 1) the long first latency at -35 mV (30); 2) the small and equivalent conductance in Ba2+ and Ca2+ (32, 41); and 3) complete voltage-dependent inactivation at -40 mV (13, 29) is consistent with the documented behavior of T-type Ca2+ channels.


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Fig. 1.   Characteristics of unitary T-type Ca2+ channel currents. Consecutive cell-attached recordings from T-type Ca2+ channels. A: effect of holding potential on single-channel openings. Voltage protocol is shown above current records. Currents were sampled at 12.5 kHz and filtered at 1 kHz. Data show potential-dependent inactivation. B: amplitude histogram of single-channel openings. Detected events fitted to 1 Gaussian distribution (see METHODS) defining a conductance level of 0.51 ± 0.061 pA. C: unitary current-voltage relationship. Data obtained from at least 4 patches at each test potential (5 measurements each) using 110 BaCl2 () or 110 CaCl2 (open circle ) as the charge carrier. Straight line is linear regression fit of BaCl2 data yielding slope conductance of 8.7 pS (r = 0.99).

Effect of extracellular Ca2+ elevation on LVA Ca2+ channels. Exposure to 140 mM extracellular K+ reduces membrane potential to zero (16) and allows intracellular Ca2+ levels to be altered by changes in extracellular Ca2+ (18). In AG cells, half-maximal activation of CaMKII is attained by 1.5 µM Ca2+ at a saturating concentration of CaM (700 nM) (10). LVA channel activity was examined with extracellular Ca2+ set to 150 nM and 1.0 µM using Ca2+-EGTA buffers. With the use of these buffers, [Ca2+]i in the bulk cytosol rose from 62 ± 3 nM, a value similar to that observed in unstimulated AG cells incubated in physiological KRB (3.5 mM K+), to 473 ± 41 nM (mean ± SE, n = 10), a value similar to that attained on 5 mM K+ depolarization (10).

The recordings illustrated in Fig. 2 were typical of most cell-attached patches, and visual inspection shows that elevation of extracellular Ca2+ induced an increase in T-type Ca2+ channel activity. As illustrated in Fig. 2A, at low external Ca2+, small conductance openings evoked by depolarizing pulses to -35 mV were generally brief and infrequent. Because of this low frequency of channel opening, channel kinetics were analyzed from records compiled from 10 cells that had only a single channel appearing to be active in 100 measured consecutive sweeps. At -35 mV with 110 mM Ba2+ as the charge carrier, channels opened with a first latency of 68 ± 2 ms (mean ± SE), a frequency of 8.8 ± 1.7 openings/s and an amplitude of 0.51 ± 0.06 pA. Shown at the bottom of the current records, an ensemble average of 1,000 sweeps displayed a peak current amplitude of 0.023 pA. By contrast, elevation of extracellular Ca2+ to 1.0 µM evoked a greater than threefold increase in the mean frequency of opening to 30.8 ± 4.7 openings/s (P <=  0.05) and an ~30% reduction in first latency to 48 ±1 ms (P <=  0.05). Whereas the main single-channel amplitude observed in the presence of elevated Ca2+ was similar to that observed in low Ca2+, a subconductance state was also identified in the records (see Fig. 2A). The resulting amplitude histogram was well fitted with the sum of two Gaussian distributions with mean amplitudes of the main and subconductance states equal to 0.47 pA and 0.20 pA, respectively. While subconductance states have been previously reported for LVA channels (8, 36), we focused subsequent analysis on the properties of the main conductance state that constituted >93% of the detected events. The ensemble averaged current after extracellular Ca2+ elevation attained a peak current of 0.048 pA, a value that was approximately twofold greater than that observed in low extracellular Ca2+. Although this averaged current displayed little channel inactivation during the 150-ms test pulse to -35 mV, channel inactivation was complete within the 6 s of interpulse holding at -40 mV (see Fig. 1A). Very slow inactivation of LVA channels during weak test depolarizations has been previously observed (11, 17, 30).


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Fig. 2.   Effect of extracellular Ca2+ on unitary Ca2+ channel currents. A: cell-attached recordings selected to show diverse patterns of openings elicited upon raising extracellular Ca2+ from 150 nM to 1.0 µM in 140 mM K+-depolarized cells. Traces below the single-channel recordings are the average current obtained from 100 consecutive sweeps from each of 10 cells. B: channel probability of opening (NPo) analysis from same patches recorded in A. Data show consistent increase in NPo evoked by 1.0 µM Ca2+ (Ca2+-dependent potentiation).

Figure 2B shows that elevation of extracellular Ca2+ consistently increased channel activity. Plotted is the percent open time calculated from 100 consecutive sweeps for each of 10 cells before and after Ca2+ was elevated. Although there is considerable variation in the NPo among control cells, Ca2+ increased NPo in all cell-attached patches from 200 to 600%. This increase in NPo was primarily the consequence of a reduction in the number of blank sweeps (557-245 blanks/1,000 sweeps, P < 0.001) and an increase in the number of openings in nonblank sweeps (3.4 ± 0.6 to 6.5 ± 0.8 openings/sweep; P <=  0.05). An analysis of NPo by sweep indicated a continuous range of NPo values in low and high extracellular Ca2+ rather than values that grouped near a few distinct values. Thus unlike N-type Ca2+ channels that are induced by epinephrine to switch gating modes, Ca2+ elevation does not appear to increase channel activity by altering the modal behavior of the T-type Ca2+ channel (7). Figure 3 shows a log-log plot of an open-state dwell-time distribution that was well fit to the sum of two exponentials (r > 0.98), suggesting the existence of two open states with open-time constants that differed from each other by nearly an order of magnitude (tau 1 = 0.31 ± 0.04 ms; tau 2 = 2.29 ± 0.64 ms). In low extracellular Ca2+, ~62% of the detected events (1,354/1,000 sweeps) were identified by the fast open-time constant with the remainder characterized by the slower time constant. Raising the bath Ca2+ concentration increased the number of detected openings to 4,734 (per 1,000 sweeps) but neither prolonged the fast or slow open-time constants (tau 1 = 0.26 ± 0.02 ms; tau 2 = 2.21 ± 0.35 ms) nor increased the relative proportion of openings identified by the slow open-time constant (70%: tau 1; 30%: tau 2). Thus the Ca2+-induced increase in NPo could not be explained by a prolongation of the duration of open events. Rather, the present data suggested that the Ca2+-induced increase in NPo was associated with a greater than threefold increase in the number of openings assigned to each open-state distribution.


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Fig. 3.   Effect of extracellular Ca2+ on dwell-time distributions. Log-log histograms of linearly binned open times obtained with a digital sampling interval of 0.08 ms. The best-fit 2 exponential is shown superimposed with components. A: at 150 nM, extracellular Ca2+tau 01 (fast) = 0.3 ± 0.01 ms, events/bin = 360 ± 45; tau 02 (slow)= 2.3 ± 0.6 ms, events/bin = 30 ± 10; r = 0.95. B: at 1.0 µM, extracellular Ca2+tau 01 (fast) = 0.3 ± 0.01 ms, events/bin = 1,855 ± 129, r = 0.97; tau 02 (slow) = 2.2 ± 0.6 ms, events/bin = 95 ± 18, r = 0.98. Data show Ca2+ elevation increases frequency of fast and slow events.

Visual inspection of the single-channel records also showed that many of the events did not occur in isolation but were separated by short- and long-lived closures, a characteristic of bursting activity. As expected, the distribution of dwell times in the closed state was multiexponential. However, because the Po of the channel was low in both low and high Ca2+ and the last closing could not be distinguished from inactivation, many long-duration channel closures were missed and could not be adequately quantified. Nevertheless, a Ca2+-induced reduction in a slow closed/inactivated state time constant or proportion of closed/inactivated state events would be expected to underlie the observed Ca2+-induced increase in the number of openings in nonblank sweeps. We examined records for burst activity with the use of critical closed-time estimates based on criteria by Colquhoun and Sigworth (5a) (2.4 and 2.0 ms: low and high Ca2+). With the use of these estimates, only 6.5% of the events would be expected to be misclassified. As anticipated from the previous kinetic analysis of unitary events, high Ca2+ exposure increased the observed number of bursts from 534 to 1,741. This increase in burst number was not accompanied by an increase in the mean number of openings/burst (1.8 ± 1.7: low Ca2+; 2.2 ± 2.2: high Ca2+) or a change in the distribution of burst lengths. Thus whether analyzed as unitary or bursting events, Ca2+ increased T-type Ca2+ channel activity by effecting an increase in event frequency rather than by prolonging the length of events.

Mediation by Ca2+/CaM-dependent protein kinase II. In the next series of experiments, we determined whether CaMKII activity underlies the potentiating effect of extracellular Ca2+ elevation. Previous studies from our laboratory have shown that in AG cells, activation of CaMKII induces a hyperpolarizing shift in the voltage dependence of activation of the T-type Ca2+ channel (27). An increase in event frequency is consistent with the CaMKII-evoked shift in the voltage dependence of activation of whole cell currents that occurs without a change in the tau  of deactivation of whole cell T-type tail currents. Figure 4 shows that inhibition of CaMKII activity prevents the potentiating effect of extracellular Ca2+ on single-channel activity in cell-attached patches. CaMKII activation was blocked by preincubation of AG cells with KN-62 (3 µM, 1 h), a lipophilic inhibitor that competes with CaM for its binding site on the kinase. Representative single-channel recordings from AG cells treated with KN-62 (Fig. 4A) showed that KN-62 did not prevent Ca2+ channel openings elicited on depolarization to -35 mV in low Ca2+ but blocked the elevation in channel activity by high extracellular Ca2+. In untreated cells, an analysis of the Po indicated that 1.0 µM Ca2+ increased NPo 4.2-fold from 1.4 ± 0.3 to 5.9 ± 0.9% (P <=  0.05) in 12 cell-attached patches. This increase in NPo compared with an insignificant effect in cells treated with KN-62 [2.07 ± 0.3 vs. 2.6 ± 0.5%; n = 8; not significant (n.s.)]. By contrast, pretreatment with the inactive analog of KN-62, KN-04, which differs from KN-62 by only a single carbonyl residue, did not prevent the increase in channel activity (Fig. 4A) evoked by Ca2+ (2.0 ± 0.8 vs. 6.2 ± 1.4%; n = 7; P < 0.05). These data suggested that the potentiating effect of Ca2+ on LVA Ca2+ channel opening depended on CaMKII activation.


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Fig. 4.   Effect of Ca2+/calmodulin-dependent protein kinase (CaMKII) inhibition on Ca2+-dependent potentiation. A: cell-attached recordings selected to show the effect of Ca2+ elevation when CaMKII activation is inhibited (KN-62) or preserved (KN-04). Voltage protocol is shown above current records. B: NPo analysis of cells bathed in extracellular Ca2+ of 150 nM or 1.0 µM Ca2+ that were untreated or pretreated with KN-62 (active analog) or KN-04 (inactive analog). Data show active analog selectively inhibits potentiated Ca2+ channel activity.

The role of CaMKII in the regulation of LVA Ca2+ channels was further investigated using excised inside-out patches. Unlike high voltage-activated (HVA) channels where activity ceases within minutes of patch excision, T-type Ca2+ channels can remain active in this configuration (3, 22). Because excision into low Ca2+ (150-nM free) increased baseline channel activity to submaximal stimulatory levels, bath Ca2+ was reduced to 50 nM, close to the measured intracellular Ca2+ level. This observed "run up" of single-channel activity on patch excision into 150-nM free Ca2+ confirms the disequilibrium that exists between extracellular Ca2+ and intracellular Ca2+ in the cell-attached configuration. In the presence of 1 mM ATP, 200 µM GTP, and 0.2 µM CaM, LVA channel activity persisted during the entire 20-min recording period. Depolarization to -35 mV evoked unitary currents of small amplitude that were not different from those recorded in the cell-attached configuration (Fig. 5). Elevation of Ca2+ to 500 nM greatly increased Ca2+ channel activity. NPo rose 3.8-fold from 0.8 ± 0.2 to 3.0 ± 0.8% (n = 10, P <=  0.05) upon Ca2+ stimulation, demonstrating that Ca2+-induced potentiation can be reconstituted in the membrane patch. In the excised configuration, the highly specific, nonpermeant CaMKII inhibitor, autocamtide 2-related inhibitory peptide (AIP) (20), could be used to reduce kinase activity. This synthetic peptide (KKALRRQEAVDAL) is modeled on the autonomy site (Thr-286) in the autoregulatory domain (RQETV) of CaMKIIalpha but has an Ala substitution for Thr (15). Inclusion of this peptide in the external solution at 1 µM [a concentration ~25 times the IC50 for CaMKII inhibition (20)] prevented the increase in Ca2+ channel activity elicited by Ca2+ elevation (0.8 ± 0.2 vs. 0.9 ± 0.2; n = 11; n.s.). The observed inhibition of LVA channel activity induced by AIP was specific to stimulated activity since this inhibitor of the kinase did not reduce basal Ca2+ channel activity recorded in 50 nM Ca2+. Together, these data suggest that CaMKII is contained within the domain of the excised patch, that regulation occurred locally, and that the participation of a distant kinase cascade is not obligatory.


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Fig. 5.   Effect of CaMKII activation or inhibition on Ca2+ channel activity in the excised patch. A: excised-patch recordings selected to show effect of Ca2+ elevation (50-500 nM) and CaMKII inhibition effected by autocamtide 2-related inhibitory peptide (AIP). Voltage protocol is shown above records. B: NPo analysis of recordings from inside-out patches bathed in 50 nM or 500 nM with or without 1 µM AIP. Data show selective inhibition of CaMKII blocks Ca2+ from potentiating channel activity.

Although our studies to this point indicated the importance of CaMKII activation in the regulation of T-type Ca2+channel activity, they did not rule out the participation of other Ca2+-dependent effectors. To address this concern, the efficacy of preactivated CaMKII on single-channel activity was assessed in the excised patch in the absence of Ca2+ elevation. We used mutant recombinant CaMKIIalpha , in which residue 286, the threonine phosphorylation site, was replaced by an aspartate (T286D-CaMKIIalpha ). This substitution produces the same functional effect on the kinase as the autophosphorylation of Thr-286 in the autoinhibitory domain and generates partial activity in the absence of activation by CaM and Ca2+ (see METHODS) by increasing the affinity of CaMKII for CaM by ~100-fold (31) and permitting Ca2+-independent (autonomous) phosphorylation of substrates following dissociation of Ca2+/CaM (reviewed in Ref. 1). While keeping bath Ca2+ fixed at 50 nM, Ca2+ channel activity was recorded before and after the addition of T286D-CaMKII (~60 nM final). As illustrated in the representative recordings, single-channel activity was greatly increased by the addition of T286D-CaMKII (Fig. 6A). Within 5 min of kinase exposure, NPo rose 2.8-fold from a mean value of 0.5 ± 0.1 to 1.4 ± 0.2% (n = 9, P <=  0.05). This effect was dependent on ATP (ATP free: 0.38 ±0.2; n = 6; n.s.) and specific to the addition of active kinase because neither the addition of vehicle (0.4 ± 0.1; n = 5; n.s.) nor heat-inactivated kinase (0.6 ± 0.2; n = 5; n.s.) were effective in increasing NPo. As expected, subsequent addition of noninactivated kinase and ATP resulted in enhanced channel activity. Thus CaMKII activity was necessary and sufficient to increase NPo in the absence of activation of other Ca2+-dependent effectors.


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Fig. 6.   Effect of constitutively active CaMKII on Ca2+ channel activity. Asp replaced Thr-286 in recombinant CaMKIIalpha , rendering the kinase active in the absence of Ca2+ elevation. A: excised-patch recordings selected to show effect of active or heat-inactivated T286D-CaMKII (~60 nM) in 50 nM Ca2+. Voltage protocol is shown above records. B: NPo analysis of recordings taken before and 1-5 min after the addition of T286D-CaMKII, vehicle, or heat-inactivated T286D-CaMKII. * P < 0.5 vs. T1 activity. Data show T286D-CaMKII is sufficient to increase channel activity.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ca2+-dependent potentiation. The modulation of Ca2+ channel currents by Ca2+ has been documented for at least two major types of HVA Ca2+ channels (L-type, P/Q-type) (9, 12, 14, 18, 25, 28, 48, 50). Here, we describe at the single-channel level potentiation of LVA Ca2+ channel currents by Ca2+. With Ca2+ or Ba2+ as the permeant ion, elevation of bath Ca2+ increased LVA Ca2+ channel Po in K+-depolarized cell-attached patches and excised patches in the inside-out configuration. Potentiation of LVA channel activity by Ca2+ contrasts with the well-known effect of Ca2+ to inactivate L-type and P/Q-type Ca2+ channels, but it mimics the less familiar effect of Ca2+ to potentiate L-type Ca2+ channel activity (18, 28, 48). In the present study, a biphasic effect of Ca2+ on the activity of LVA channels was not observed. We report that Ca2+ increased Ca2+ channel Po by 200-600% primarily as a result of 1) an evoked increase in channel availability, as indicated by the reduction in null sweeps, and 2) by an elicited increase in the conditional probability of opening, as judged by an increase in the Po of nonblank sweeps with only a small change in first latency. In contrast, the characteristics of feed-forward regulation of L-type Ca2+ channels by Ca2+ include both an increase in the frequency of channel opening and an additional, but modest, increase in the duration of the open state (18). In the present study, the Ca2+-dependent enhancement of T-type channels was not accompanied by a change in the conductance of the channel or by a prolongation of either open state but, rather, by an increase in the frequency of channel opening. The mechanism by which the frequency of channel opening is achieved may involve the modulation of one or more closed or inactivated states that can precede channel opening, as recently described by Serrano et al. (40). Thus a detailed kinetic description underlying this effect must await further studies.

LVA channels in AG cells exhibited a conductance of 8.7 pS, a unitary current amplitude of -0.51 pA at -35 mV, and slow but full steady-state potential-dependent inactivation negative to -35 mV. LVA Ca2+ channels characterized in glomerulosa cells, cardiac myocytes, and neurons have similar properties measured with comparable solutions (3, 11, 13, 29, 30, 32, 33). At least two groups of open times could be distinguished: one characterized by a fast (0.3 ms) and the other a slower (2.2, 2.3) open-state time constant. Our finding of a bimodal distribution of open times, which is suggestive of two open states, has not been uniformly observed for the T-type Ca2+ channel (3, 4, 8, 13). While it is conceivable that the algorithm used to detect channel openings permitted greater sensitivity, it is also possible that the variable observation of two open states may relate to heterogeneity within the T-type Ca2+ channel gene family and/or posttranslational modifications. Since the cloning of the alpha 1G (36), two new genes encoding T-type Ca2+ channels have been identified (alpha 1H and alpha 1I) that share 90-80% sequence identity across putative transmembrane segments (6). Although at the whole cell level these channel paralogs have been shown to have very similar voltage-dependent properties (24), to date characterization at the single-channel level has been minimal (6, 24, 35, 36, 46). Therefore, it remains possible that the single-channel behavior reported here may be unique to a subtype within the T-type Ca2+ channel family.

Modulation by CaMKII. In the cell-attached patch, we observed that Ca2+-dependent potentiation was prevented by pretreatment with KN-62. This inhibitor of the CaM-kinase family competes with Ca2+/CaM for binding and thus displays a more selective inhibitory activity than drugs like the trifluoperazines that bind directly to CaM and thereby inhibit all CaM-dependent enzymes (21). Nonetheless, similarities in the CaM binding site among CaM-dependent kinases explains the reported inhibition of both CaMKII and CaMKIV by KN-62 (20). Thus, despite the fact that the inactive analog of KN-62, KN-04, was without effect, the inhibition of Ca2+-dependent potentiation by KN-62 cannot be interpreted as strong evidence for the unique participation of CaMKII.

In the excised configuration, Ca2+-dependent potentiation was inhibited by AIP, a peptide that interacts at two distinct substrate binding sites on CaMKII. AIP competes noncompetitively with exogenous substrates for phosphorylation and competitively with the endogenous substrate, the autophosphorylation domain of the kinase (19). As such, AIP inhibited CaMKII selectively in the absence and presence of CaM. At 1 µM, the concentration of inhibitor used in this study, AIP completely inhibited CaMKII activity without effecting other multifunctional protein kinases such as CaMKIV, cAMP-dependent protein kinase, and protein kinase C (20). Inhibition of Ca2+-dependent potentiation by AIP in the excised patch and the duplication of Ca2+ channel activation evoked by the addition of constitutively active recombinant CaMKII (T286D-CaMKII) provide strong evidence for the regulation of T-type Ca2+ channel activity by CaMKII in the AG cell. Moreover, because Ca2+-induced potentiation of T-type Ca2+ channel activity required ATP and active kinase, this regulation differed mechanistically from the regulation of L- and P/Q-type Ca2+ channels by CaM recently reported (23, 49). In these studies, the direct binding of CaM (to motifs on the COOH-terminal tail of the Ca2+ channel subunits alpha 1C or alpha 1A) was observed to promote inactivation and facilitation of these high voltage-activated Ca2+ channels independently of kinase activation. Because in our study Ca2+ channel activation also could be evoked by T286D-CaMKII in the absence of elevated Ca2+, the participation of an additional Ca2+-dependent effector downstream of CaMKII in the regulatory cascade was ruled out. Therefore, in the AG cell, activation of CaMKII is both necessary and sufficient to initiate a feed-forward regulation of T-type Ca2+ channels by Ca2+.

The fact that modulation of LVA Ca2+ channels by Ca2+ elevation was recapitulated in inside-out excised patches implies that at least some of the AG cell CaMKII remained associated with the excised patch. This could in principal occur via the association of CaMKII with plasma membrane proteins or with lipids. Several recent studies have provided evidence for similar localization of CaMKII close to sites of Ca2+ entry. For example, neuronal CaMKII binds to the NR-2B subunit of N-methyl-D-aspartic acid (NMDA)-type glutamate receptors, a ligand and voltage-gated Ca2+ channel (42). Thus CaMKII is appropriately located to be very rapidly activated by Ca2+ entering through this receptor and, by phosphorylating NMDA receptor subunits, may regulate their function (34). Similarly, in cardiomyocytes, a portion of the CaMKII appears to colocalize with subunits of the L-type Ca2+ channel (47), where it presumably plays a role in the feedback regulation of Ca2+ entry. Thus the retention of CaMKII on excised AG cell membrane patches strongly suggests that this kinase is appropriately localized to be uniquely sensitive to Ca2+ entering through LVA Ca2+ channels and to provide rapid feedback (forward) regulation.

In the AG cell, the selective enhancement of LVA channel activity at negative potentials by CaMKII activation plays a critical role in setting the level of steady-state Ca2+ entry and the level of aldosterone secretion elicited by ANG II stimulation. An increase in single-channel activity reported here offers insight into the obligatory signaling events underlying the CaMKII-induced change in whole cell channel behavior. In chronic heart failure, sustained activation of the renin-angiotensin system contributes pathologically to Na+ retention, volume expansion, and the harmful remodeling of the heart and vasculature. In light of the very recent findings indicating that blockade of aldosterone receptors in conjunction with conventional therapies further reduces the incidence of death among patients with severe heart failure (38), targeted disruption of this regulatory mechanism may be of significant clinical utility.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-36977 (to P. Q. Barrett).


    FOOTNOTES

Address for reprint requests and other correspondence: P. Q. Barrett, Dept. of Pharmacology, Univ. of Virginia School of Medicine, 1300 Jefferson Park Ave., Charlottesville, VA 22903 (E-mail: pqb4b{at}virginia.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.

Received 6 January 2000; accepted in final form 17 July 2000.


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