Intracellular Ca²⁺ Inhibits Smooth Muscle L-Type Ca²⁺ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Abstract

Modulation of L-type Ca²⁺ channels by tonic elevation of cytoplasmic Ca²⁺ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba²⁺ was used as charge carrier, and run down of Ca²⁺ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca²⁺ in intact cells by elevation of extracellular Ca²⁺ in the presence of the ionophore A23187 inhibited the activity of L-type Ca²⁺ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca²⁺ with fura-2 revealed a 50% inhibitory concentration (IC₅₀) of 260 nM and a Hill coefficient close to 4 for Ca²⁺- dependent inhibition. Ca²⁺-induced inhibition of Ca²⁺ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca²⁺-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca²⁺ at the cytoplasmic side of inside-out patches inhibited Ca²⁺ channels with an IC₅₀ of 2 μM and a Hill coefficient close to unity. Direct Ca²⁺-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca²⁺ concentration of 1 μM inhibited Ca²⁺ channel open probability and availability. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC₅₀ of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca²⁺ sensitivity of Ca²⁺ channels in intact cells. Our results suggest that L-type Ca²⁺ channels of smooth muscle are controlled by two Ca²⁺-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca²⁺ with a single membrane-associated site that may reside on the channel protein itself.

introduction

The function of L-type Ca²⁺ channels is controlled by voltage as well as by the Ca²⁺ influx through the channel (Kass and Sanguinetti, 1984; Eckert and Chad, 1984; Lee et al., 1985). An inactivation process is induced by the charge carrier Ca²⁺ itself, providing a negative feedback mechanism that is considered crucial for Ca²⁺ homeostasis in a variety of cell types, including smooth muscle cells (Jmari et al., 1986; Granitkevich et al., 1987; Granitkevich and Isenberg, 1991). There is an ongoing debate as to whether Ca²⁺ induces channel inactivation via direct binding to proteins of the channel complex or via a more remote mechanism such as Ca²⁺-dependent dephosphorylation (Chad and Eckert, 1986; Armstrong, 1989) or disruption of the cells' cytoskeleton (Johnson and Byerly, 1993). Inhibition of L-type channels by cytosolic Ca²⁺ has been demonstrated in cell-free membranes, supporting the view of a rather direct inhibitory mechanism such as interaction of Ca²⁺ with the channel (Romanin et al., 1992; Haack and Rosenberg, 1994; Schmid et al., 1995). Recent evidence suggests that Ca²⁺-induced inactivation is a property of the pore-forming α1 subunit of the Ca²⁺ channel (Zong and Hofmann, 1996), and an EF-hand Ca²⁺ binding motif in the α1 subunit has recently been identified as a structure that is essential for Ca²⁺-dependent inactivation (De Leon et al., 1995). Nonetheless, different lines of investigations (Ohya et al., 1988; Hirano and Hiraoka, 1994; You et al., 1995) support the hypothesis of a more complex Ca²⁺-dependent control of L-type Ca²⁺ channels, involving Ca²⁺-dependent enzymatic mechanisms in addition to direct binding of Ca²⁺ to the channel. Both stimulatory (Hirano and Hiraoka, 1994) and inhibitory modulation (Ohya et al., 1988; Hirano and Hiraoka, 1994; You et al., 1995) of Ca²⁺ channels has been observed in response to increases in bulk cytoplasmic Ca²⁺ to levels lower than the concentrations reported for inhibition of L-type Ca²⁺ channels in cell-free membranes (Romanin et al., 1992; Haack and Rosenberg, 1994; Schmid et al., 1995). Candidate mechanisms for a remote control of L-type Ca²⁺ channels in cardiac and smooth muscle are Ca²⁺-dependent phosphorylation and dephosphorylation (Chad and Eckert, 1986; Armstrong, 1989; Hirano and Hiraoka, 1994), as well as Ca²⁺-dependent disruption of the cytoskeleton (Johnson and Byerly, 1993, 1994).

The present study was aimed at comparing Ca²⁺- dependent modulation of smooth muscle L-type Ca²⁺ channels in intact cells and cell-free patches and to test for a possible role of phosphorylation/dephosphorylation and modification of the cytoskeleton in intact cells. We present evidence for the control of L-type Ca²⁺ channels in vascular smooth muscle cells by two Ca²⁺-dependent mechanisms that comprise both dephosphorylation by protein phosphatase 2B and direct binding of Ca²⁺ to a membrane-associated site.

materials and methods

Cell Preparation

The media of human umbilical veins was enzymatically disaggregated to obtain single smooth muscle cells as described previously (Schuhmann and Groschner, 1994). In brief, endothelial cells were removed with dispase (type II; Boehringer Mannheim, Mannheim, Germany), and smooth muscle tissue was subsequently dissociated by filling the vessels with low Ca²⁺ (0.1 mM) Hanks buffer (Sera Lab Ltd., Sussex, UK), supplemented with 0.5 mg/ml collagenase (type II; Worthington Biochemical Corp., Freehold, NJ), 0.5 mg/ml trypsin inhibitor (Worthington Biochemical Corp.), and 1 mg/ml fatty acid–free bovine serum albumin. Vessels filled with collagenase containing Hanks solution were incubated at 37°C. After incubation for 10 min, the Hanks buffer contained single, mostly relaxed, elongated smooth muscle cells that were harvested by centrifugation (5 min, 250 g). The cells were resuspended and stored in a solution containing (mM): 110 K⁺ aspartate, 20 KCl, 2 MgCl₂, 20 HEPES, 2 EGTA (pCa = 7, see below) at 4°C, and used for experimentation within 36 h.

Measurement of Single-channel Currents

Cell potentials were set to approximately zero by use of a high K⁺ low Cl⁻ extracellular solution that contained (mM): 110 K⁺ aspartate, 20 KCl, 2 MgCl₂, 20 HEPES, 2 EGTA, pH was adjusted to 7.4 with N -methyl-d-glucamine, and pCa was adjusted using a Ca²⁺-sensitive electrode. Patch pipettes were fabricated from borosilicate glass (Clark Electromedical Instruments, Pangbourne, UK), and had resistances of 5–10 MOhm. For recording of Ba²⁺ currents through single Ca²⁺ channels, the pipettes were filled with a solution containing (mM): 10 BaCl₂, 100 NaCl, 30 TEA-Cl, and 15 HEPES, pH adjusted to 7.4. The dihydropyridine-Ca²⁺ channel activator S(−)-BayK 8644 (0.5 μM) was included in the pipette solution to facilitate stabilization of channel activity in inside-out patches. Run down of Ca²⁺ channel activity in inside-out experiments was prevented by addition of calpastatin (2 U/ml) plus 1 mM ATP/Na₂ to the bath solution before patch excision (Romanin et al., 1992). In experiments recording Ca²⁺-activated K⁺ channels, the pipette solution contained (mM): 137 NaCl, 5 KCl, 2.5 CaCl₂, 2 MgCl₂, 10 HEPES, pH was adjusted to 7.4. All experiments were performed at room temperature. Exchange of bath solutions and administration of drugs was performed during constant flow perfusion.

Voltage clamp and current amplification was performed with a patch-clamp amplifier (EPC/7; List, Darmstadt, Germany). Current records were filtered at 1 kHz (−3 dB) and digitized at a rate of 5 kHz. Experiments were controlled using pClamp software (Axon Instruments, Foster City, CA). For idealization of current records, a custom-made level detection software was employed (Pastushenko and Schindler, 1997). Ca²⁺ channel activity was calculated as the mean number of open channels during depolarizing pulses. The dependency of channel activity on cytoplasmic free Ca²⁺ concentration was characterized in terms of 50% inhibitory concentration (IC₅₀ values)¹ and Hill coefficients by fitting the data with a four-parameter logistic function (De Lean et al., 1978). The gating properties of single channels were analyzed in terms of channel availability P _s; i.e., the probability that a channel will open upon depolarization, as well as the open probability of available channels as outlined below.

Determination of Availability and Open Probability

Recently, a method was developed (Schmid et al., 1995; Baumgartner et al., 1997) to allow for an estimation of the number of channels, their availability P _s and open probability P _o from idealized channel traces with n < 9 channels based on the following assumptions: (a) the channels are identical and behave independently; (b) the total number of channels remains constant within the duration of the recording; and (c) the process regulating channels' gating within a sweep is distribution ergodic with respect to the number of sampling points in each conductance level. Hence, the probability of finding a sampling point in the k ^th conductance level can be described by a binomial distribution with the parameters n _A and P _o.

1

Eq. 1 and assumption c allow us to determine the probability for one sweep (Eq. 2) at given n _A with the following variables defined as: n, maximum number of channels in a patch; n _Ai, available channels in the i  ^th sweep; t _ik, number of sampling points in the k ^th conductance level in the i ^th sweep; T _i, vector of t _ik for the i  ^th sweep; T, matrix of all t _ik; T _ges, number of sampling points in a sweep; P _s, availability; P _o, open probability; M, total number of sweeps; K _i, set of conductance levels that occur in the i ^th sweep.

The elements of T _i that describe the i ^th sweep obey a multinomial distribution (Weiß, 1987) and the probability of T _i at given n _Ai and P _o is

2

if n _Ai is higher than or equal to the highest conductance level in T _i. If n _Ai is smaller than the highest conductance level, then

3

Because of assumptions a and b, the n _Ai are binomially distributed, yielding the probability for T _i at given n, P _S, and P _o to be

4

leading to the probability for T comprising all sweeps

5

Using Eq. 5, a maximum likelihood estimator for n, P _S, and P _o was constructed by maximizing the probability for T.

Measurement of Intracellular Concentrations of Ca²⁺ and H⁺

Cytoplasmic free Ca²⁺([Ca²⁺]_i) and intracellular pH (pH_i) were determined using the fluorescent Ca²⁺ and pH indicators fura-2 and BCECF ((2′,7′)-bis(carboxyethyl)-(5,6)-carboxyfluorescein), as described by Wakabayashi and Groschner (1996). In brief, cells were suspended in 5 ml of physiological solution containing (mM): 137 NaCl, 5 KCl, 2.5 CaCl₂, 2 MgCl₂, 10 HEPES, pH 7.4, and loaded with fura-2 or BCECF for 60 min at 37°C. After loading, the cells were once washed and resuspended in high K⁺ low Cl⁻ extracellular solution (see above). Fluorescence measurements were carried out using a dual wavelength spectrophotofluorometer (F2000; Hitachi Ltd., Tokyo, Japan). The cell suspension (2 ml) was stirred and maintained at room temperature. For [Ca²⁺]_i measurement, excitation wavelengths were 340 and 380 nm, and emission was collected at 510 nm. For pH_i measurement, excitation wavelengths were 506 and 455 nm, and emission was collected at 530 nm. Using the ratios (R) of fluorescence intensity (F), F₃₄₀/F₃₈₀ and F₅₀₆/F₄₅₅, the fractional changes in [Ca²⁺]_i and pH_i were determined, respectively. Fluorescence after sequential addition of 0.1% Triton X-100 and EGTA to the cell suspension provided the respective maximum fluorescence ratio (R_max) and minimum fluorescence ratio (R_min). [Ca²⁺]_i was calculated as described (Wakabayashi and Groschner, 1996). Calibration of pH_i measurements were performed using nigericin (7 μM)-containing high K⁺ solution at various extracellular pH_i values (pH_o).

Assay of p-Nitrophenyl Phosphate Phosphatase Activity

The activity of protein phosphatase 2B was measured employing p-nitrophenyl phosphate (pNPP) as substrate as described (Takai and Mieskes, 1991). 1 U phosphatase activity was the amount that catalyzed dephosphorylation of 1 mmol pNPP/min.

Statistics

Averaged data are given as mean ± SEM from the indicated number of experiments. Statistical analysis was performed using Student's t test for unpaired values. Differences were considered statistically significant at P < 0.05.

Materials

PP2B was obtained from Upstate Biotechnology Inc. (Lake Placid, NY), collagenase, type CLS II, and soybean trypsin inhibitor were obtained from Worthington Biochemical Corp., dispase type II was from Boehringer Mannheim, fatty acid–free bovine serum albumin was from Behring (Marburg, Germany), Hanks balanced salt solution was from Sera Lab Ltd., H-7, cytochalasin B, and cyclosporin A were from Research Biochemicals, Inc. (Natic, MA), and calpastatin and all other chemicals were from Sigma Chemical Co. (Deisenhofen, Germany). Calpastatin was dialyzed overnight against bath solutions (high K⁺ low Cl⁻ solutions, see above).

results

Ca²⁺-dependent Inhibition of L-Type Ca²⁺ Channels in Intact Cells

Ca²⁺-dependent modulation of L-type Ca²⁺ channels in intact cells was studied by raising intracelluar Ca²⁺ of the cells via elevation of extracellular Ca²⁺ in the presence of the Ca²⁺ ionophore A23187 (1 μM). A typical experiment is illustrated in Fig. 1. The cell was initially bathed in a solution containing ∼10 nM free Ca²⁺ (pCa 8). A23187 by itself did not affect channel activity in the cell-attached patch under these conditions. Extracellular Ca²⁺ was increased in the presence of A23187 to 10 μM, and subsequently to 100 μM. Channel activity was barely effected at 10 μM extracellular Ca²⁺, but clearly suppressed when Ca²⁺ of the bath solution was raised to 100 μM, and activity recovered partially during a following period of reduction of extracellular Ca²⁺. The actual level of average cytoplasmic free Ca²⁺ ([Ca²⁺]_i) obtained during elevation of extracellular Ca²⁺ was measured in parallel experiments using the Ca²⁺-sensitive fluorescent dye fura-2. As shown in Fig. 2 A, [Ca²⁺]_i increased barely upon application of the Ca²⁺ ionophore at 10 nM free extracellular Ca²⁺; i.e., an extracellular pCa (pCa_o) of 8. As expected from the ability of A23187 to deplete intracellular Ca²⁺ stores, a small and transient rise in [Ca²⁺]_i was observed occasionally. During elevation of extracellular Ca²⁺ to 10 μM, [Ca²⁺]_i increased rapidly from a resting level of ∼50 nM to 216 ± 19 nM (n = 7). Upon further elevation of extracellular Ca²⁺ to 100 μM, [Ca²⁺]_i increased to a level of 326 ± 14 nM (∼pCa_i 6.5, n = 7). These values of [Ca²⁺]_i did not change significantly within a period of 2–4 min after elevation of extracellular Ca²⁺. To obtain additional information on the actual levels of [Ca²⁺]_i at the cytoplasmic face of the plasma membrane of single cells, we measured the activity of large conductance Ca²⁺-activated (“maxi”) K⁺ channels, which are known to exhibit a typical Ca²⁺ dependence in the low micromolar range. Fig. 2 B shows a representative recording of maxi-K⁺ channel activity under conditions corresponding to those of the Ca²⁺ channel recordings illustrated in Fig. 1. Fig. 2 B (left) illustrates K⁺ channel activity in a cell-attached patch recorded at pCa_o 8 (top) and pCa_o 4 (bottom) in the presence of 1 μM A23187. It is clearly evident that channel activity increased only slightly when extracellular Ca²⁺ was raised to 100 μM (pCa_o 4). Fig. 2 B (right) illustrates the Ca²⁺ sensitivity of this K⁺ channel in the inside-out configuration. Channel activity is shown after excision of the patch into the bath solution of pCa 4. At the cytoplasmic side, this Ca²⁺ concentration caused full activation of the K⁺ channels. Reduction of the free Ca²⁺ concentration at the cytoplasmic side to 300 nM (pCa_i 6.5) diminished channel activity to a level comparable with that recorded in the cell-attached configuration at elevated extracellular Ca²⁺ (pCa_o 4, Fig. 2 B, left). In all of three experiments, the activity of maxi-K⁺ channels recorded at pCa_i 6.5 in inside-out patches was equal to or even somewhat higher than the activity observed in intact cells during a challenge with elevated Ca²⁺ (pCa_o 4) in the presence of A23187. Thus, the measurement of maxi-K⁺ channel activity in single cells was consistent with our determination of Ca_i by measurement of fura-2 fluorescence, confirming that Ca²⁺-dependent inhibition of L-type Ca²⁺ channels in intact cells indeed occurred at a level of [Ca²⁺]_i as low as ∼300 nM. Fig. 3 A shows the concentration dependence obtained for Ca²⁺-induced inhibition of L-type Ca²⁺ channels in intact cells using the [Ca²⁺]_i values determined with fura-2. The IC₅₀ value was 260 nM and a Hill coefficient (n_H) of ∼4 (3.9) was calculated.

Figure 1.

Elevation of extracellular Ca²⁺ in the presence of A23187 inhibits the activity of L-type Ca²⁺ channels in cell-attached patches. Time course of Ca²⁺ channel activity recorded in a cell- attached patch given as the mean number of open channels during individual depolarizing voltage pulses (holding potential −70 mV, test potential −20 mV, duration 500 ms, rate 0.66 Hz). Administration of 1 μM A23187 and changes in the extracellular Ca²⁺ concentration, given as pCa_o values, are indicated. (bottom) Individual current responses to depolarizing voltage pulses at the times indicated. Records were filtered at 500 Hz and digitized at 2 kHz. Closed state is indicated by arrows.

Figure 2.

Determination of intracellular Ca²⁺ levels during elevation of extracellular Ca²⁺ in the presence of A23187. (A) Time course of intracellular free Ca²⁺ ([Ca²⁺]_i) during elevation of extracellular Ca²⁺ in the presence of 1 μM A23187 measured by fura-2 fluorescence. Administration of 1 μM A23187 and changes in the extracellular Ca²⁺ concentration, given as pCa_o values, are indicated. Mean values ± SEM (n = 7) of [Ca²⁺]_i determined for the period of 2–4 min after elevation of extracellular Ca²⁺ to the respective value, are shown for pCa_o 5 and 4. (B, left) Activity of Ca²⁺-activated K⁺ channels in a cell-attached patch (test potential = 0 mV) during elevation of extracellular Ca²⁺, given as pCa_o values, in the presence of 1 μM A23187. (right) Channel activity in the same patch after excision (inside-out configuration) at pCa 4 (100 μM) and pCa 6.5 (300 nM). Records were filtered at 500 Hz and digitized at 2 kHz. Unitary current levels are indicated by dashed lines.

Figure 3.

Concentration dependence of Ca²⁺-induced inhibition of Ca²⁺ channel activity in cell-attached patches. Mean values ± SEM of channel activity obtained from two to four experiments. Data were fitted by a four-parameter logistic function, and the derived Hill coefficient (n_H) is given.

Since Ca²⁺ channel activity in smooth muscle is determined by intracellular pH (Klöckner and Isenberg, 1994), we first tested whether changes in intracellular pH might account for the inhibitory effects observed in intact cells. Using BCECF as a pH indicator, we found that pH_i did not change significantly upon administration of A23187 and subsequent elevation of extracelluar Ca²⁺. A pH_i of 7.41 ± 0.16 (n = 9) in controls and 7.40 ± 0.11 (n = 4) in the presence of A23187 plus 100 μM Ca²⁺ was measured.

We have recently reported on a mechanism of Ca²⁺ channel inhibition that involves Ca²⁺-phospholipid- dependent protein kinases (Schuhmann and Groschner, 1994); thus, we tested whether the protein kinase inhibitor H-7 is able to blunt Ca²⁺-dependent inhibition of channels in intact cells. H-7 (10 μM) did not affect the inhibitory modulation in intact cells (n = 4). Similarly, cytochalasin B, a cytoskeletal disrupter, failed to suppress Ca²⁺-dependent inhibition of L-type channels (n = 3).

Cyclosporin A Prevents Ca²⁺-dependent Inhibition of Ca²⁺ Channels in Intact Cells

A role of Ca²⁺-dependent dephosphorylation has previously been implicated in Ca²⁺-induced negative feedback mechanisms (Armstrong, 1989). Thus, we tested cyclosporin A as an inhibitor of the Ca²⁺-dependent protein phosphatase 2B (calcineurin). Elevation of extracellular Ca²⁺ from 10 nM to 100 μM (pCa_o 4) in the presence of 1 μM A23187 resulted in an increase in intracellular free Ca²⁺ to ∼300 nM (pCa_i 6.5, Fig. 2) and suppressed channel activity substantially within 1 min (Fig. 4 A). However, when cells were pretreated with cyclosporin A (1 μg/ml), channel activity remained constant even during prolonged elevation of intracellular Ca²⁺ (Fig. 4 B; n = 3). Removal of cyclosporin A resulted in the expected inhibition of Ca²⁺ channel activity, which recovered upon reduction of extracellular Ca²⁺ to pCa 8 (Fig. 4 B). Cyclosporin A by itself did not affect channel activity significantly under basal conditions (n = 3). Experiments with fura-2 confirmed that elevation of [Ca²⁺]_i induced by extracellular Ca²⁺ in the presence of A23187 remained unchanged by cyclosporin A (n = 3). Thus, cyclosporin A specifically antagonized Ca²⁺-induced suppression of channel activity. These results demonstrate that cyclosporin A interferes with Ca²⁺-dependent negative feedback control of Ca²⁺ channels in intact smooth muscle cells, and indicate a role of protein phosphatase 2B, the classical target of cyclosporin A.

Figure 4.

Cyclosporin A prevents Ca²⁺-induced inhibition of Ca²⁺ channels in intact cells. Time courses of Ca²⁺ channel activity recorded in cell-attached patches. A23187 (1 μM) is present throughout the experiments. Changes in the extra- and intracellular Ca²⁺ concentration are given as pCa values (pCa_o and pCa_i). (A) Control experiment. (B) Experiment in the presence of cyclosporin A (1 μg/ml; 10 min). Channel activity is given as the mean number of open channels during individual depolarizing voltage pulses (holding potential −70 mV, test potential −20 mV, duration 500 ms, rate 0.66 Hz). (bottom) Individual current responses to depolarizing voltage pulses at the times indicated. Records were filtered at 500 Hz and digitized at 2 kHz. Closed state is indicated by arrows.

In an attempt to further characterize the cyclosporin A–sensitive effect of cytoplasmic Ca²⁺, we evaluated the effects of Ca²⁺ on open probability (P_o) and availability (P_s); i.e., the probability for a channel to open once during a depolarization. Using a recently developed procedure that allows for determination of these parameters from multichannel records (Schmid et al., 1995), we found that Ca²⁺-induced suppression of channel activity in intact cells was associated with a reduction of P_o and in addition a marked reduction of P_s. These effects were clearly antagonized by cyclosporin A (Fig. 5).

Figure 5.

Cyclosporin A prevents Ca²⁺-induced inhibition of open probability (P_o) and availability (P_s) of channels in intact cells. P_o and P_s of channels are shown under control conditions (pCa_i 8.5) and at elevated cytoplasmic Ca²⁺ (pCa_i 6.5) in the absence and presence of cyclosporin A (1 μg/ml). Mean values ± SEM (n = 4) are given. *Significant difference versus control values.

Inhibition of L-Type Ca²⁺ Channels in Cell-free Patches by Ca²⁺ and Protein Phosphatase 2B

Ca²⁺ channel activity in excised, inside-out patches remained stable over more than 30 min when the cytoplasmic side was exposed to a solution containing calpastatin (2 U/ml) and ATP (1 mM) (Romanin et al., 1992; Schmid et al., 1995). This experimental protocol allowed for investigation of the sensitivity of smooth muscle L-type Ca²⁺ channels to cytoplasmic Ca²⁺ as well as to dephosphorylation by protein phosphatase 2B. As a first step, we studied channel inhibition by Ca²⁺ itself. As illustrated in Fig. 6, Ca²⁺ channels recorded in excised patches were apparently not affected when the Ca²⁺ concentration of the solution facing the cytoplasmic side was increased to 300 nM (pCa_i 6.5), but significantly inhibited at 10 μM free Ca²⁺ (pCa_i 5). This effect was rapidly reversible upon subsequent reduction of the free Ca²⁺ concentration to 10 nM (pCa_i 8). Fig. 7 depicts the concentration dependence obtained for Ca²⁺-induced inhibition of L-type Ca²⁺ channels in excised patches. Inhibition was characterized by an IC₅₀ value of 2 μM and a Hill coefficient (n_H) close to unity (1.1).

Figure 6.

Ca²⁺-induced inhibition of Ca²⁺ channels in cell-free patches. Time course of Ca²⁺ channel activity in the cell-attached and inside-out configuration, given as the mean number of open channels during individual depolarizing voltage pulses (holding potential −70 mV, test potential −20 mV, duration 500 ms, rate 0.66 Hz). Patch excision and subsequent changes in the Ca²⁺ concentration at the cytoplasmic side (pCa_i) are indicated. (bottom) Individual current responses to depolarizing voltage pulses at the times indicated. Records were filtered at 500 Hz and digitized at 2 kHz. Closed state is indicated by arrows.

Figure 7.

Concentration dependence of Ca²⁺-induced inhibition of Ca²⁺ channel activity in excised inside-out patches. Mean values ± SEM of channel activity obtained from two to four experiments. Data were fitted by a four parameter logistic function, and the derived Hill coefficient (n_H) is given.

Since our experiments with cyclosporin A indicated a role of PP2B in Ca²⁺-dependent downregulation of Ca²⁺ channels in intact cells, the next step was to test whether purified PP2B is able to suppress Ca²⁺ channel activity in excised patches. The effects of PP2B were studied in the presence of 1 μM calmodulin. Fig. 8 illustrates that administration of purified PP2B (1 μg/ ml) to the cyctoplasmic side of Ca²⁺ channels in inside-out patches failed to affect channel activity at pCa 8, but inhibited channels at pCa 6 within 1–2 min to a level below 10% of control (n = 3). Administration of 1 μM calmodulin alone at pCa 6 did not affect channel activity (n = 3). The concentration dependence of Ca²⁺-induced channel inhibition in the presence of PP2B (1 μg/ml) is illustrated in Fig. 9. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B inhibited Ca²⁺ channel activity in inside-out patches with an IC₅₀ of 379 nM and a Hill coefficient (n_H) of ∼3 (3.1). Consistent with the idea of PP2B effects being mediated by protein dephosphorylation, PP2B phosphatase activity was enhanced by Ca²⁺ in the same range of concentrations, with half-maximal stimulation at 430 nM free Ca²⁺ and a Hill coefficient of 4.5 (n = 4). To test whether downregulation of Ca²⁺ channels by PP2B is due to an impairment of Ca²⁺ channel stabilization by calpastatin, we preincubated calpastatin for 10 min with PP2B (1 μg/ml) plus calmodulin (1 μM) at pCa 6. This preincubation did not affect the ability of calpastatin to stabilize Ca²⁺ channel activity in excised patches (n = 2).

Figure 8.

PP2B-induced inhibition of Ca²⁺ channels in cell-free patches. Time courses of Ca²⁺ channel activity recorded in inside-out patches at a pCa_i of 8 (A) and 6 (B). Addition of PP2B (1 μg/ ml) to the cytoplasmic side is indicated. Channel activity is given as the mean number of open channels during individual depolarizing voltage pulses (holding potential −70 mV, test potential −20 mV, duration 500 ms, rate 0.66 Hz). (B, bottom) Individual current responses to depolarizing voltage pulses in the absence and presence of PP2B (12 U/ml). Records were filtered at 500 Hz and digitized at 2 kHz. Closed state is indicated by arrows.

Figure 9.

Concentration dependence of Ca²⁺-induced inhibition of Ca²⁺ channel activity in excised inside-out patches and Ca²⁺-induced stimulation of PP2B phosphatase activity. Mean values ± SEM of channel activity (○; n = 3–4) and of PP2B phosphatase activity (•; n = 5) measured in parallel with pNPP as substrate. Data were fitted by four-parameter logistic functions and the derived Hill coefficient (n_H) of Ca²⁺-dependent inhibition of channel activity is given.

To further characterize Ca²⁺- and PP2B-mediated modulation of channels in excised patches, we analyzed channel function in terms of P_o and P_s. As shown in Fig. 10, elevation of cytoplasmic Ca²⁺ to 1 μM (pCa_i 6) barely reduced P_o and P_s in the absence of PP2B. Further elevation of cytoplasmic Ca²⁺ to 10 μM (pCa_i 5) inhibited channel activity mainly by suppression of P_o. Thus, direct inhibition of Ca²⁺ channels in excised patches by cytoplasmic Ca²⁺ concentrations >1 μM was different from Ca²⁺-dependent inhibition in intact cells, which was associated with a reduction of both P_o and P_s. The Ca²⁺-dependent effect of PP2B in excised patches was characterized by a substantial reduction of P_o and P_s. Thus, channel modulation by PP2B mimicked Ca²⁺-dependent inhibition in intact smooth muscle cells. These results demonstrate two types of Ca²⁺- dependent downregulation of L-type Ca²⁺ channels in smooth muscle. Elevation of intracellular Ca²⁺ by itself inhibits specifically P_o of the channel, whereas in the presence of calmodulin plus PP2B, elevation of Ca²⁺ induces suppression of both P_o and P_s.

Figure 10.

PP2B inhibits open probability (P_o) and availability (P _s) of Ca²⁺ channels in cell-free patches. P_o and P_s of channels in inside-out patches are shown at different cytoplasmic Ca²⁺ (given as pCa) in the absence or presence of PP2B (12 U/ml). Mean values ± SEM (n = 3–4) are given. *Significant difference versus control values.

discussion

The present study provides evidence for the existence of two distinct mechanisms by which intracellular Ca²⁺ governs L-type Ca²⁺ channel function in smooth muscle. These Ca²⁺-mediated mechanisms exhibit different concentration dependencies and are based on different changes in single-channel properties. Our results suggest that intracellular Ca²⁺ inhibits smooth muscle L-type Ca²⁺ channels by induction of a dephosphorylation process mediated by PP2B and by direct binding to a membrane-associated regulatory site.

Ca²⁺ Sensitivity of Smooth Muscle L-Type Ca²⁺ Channels in Intact Cells and Cell-free Patches

In the present study, Ca²⁺ channel function was measured with the charge carrier Ba²⁺, which fails to mimic the negative feedback inhibition mediated by Ca²⁺. Consequently, the use of Ba²⁺ as charge carrier allows us to determine the dependency of channel inhibition on steady state Ca²⁺ concentrations at the cytoplasmic side. It is of note that this approach does not allow us to evaluate the effects of Ca²⁺ permeation through the pore on channel function. Nonetheless, this approach allows for a detailed characterization of regulatory Ca²⁺ interaction sites at the cytoplasmic face of the membrane and of Ca²⁺-dependent intracellular regulatory mechanisms. We demonstrate that L-type Ca²⁺ channels exhibit different Ca²⁺ sensitivities in intact cells and cell-free patches. The observed Ca²⁺ sensitivity of smooth muscle channels in inside-out patches (IC₅₀ = 2 μM) is in accordance with reports on the Ca²⁺ sensitivity of cardiac L-type channels in excised patches (Romanin et al., 1992) and in planar lipid bilayers (Haack and Rosenberg, 1994). In a previous study on the Ca²⁺ sensitivity of L-type channels in excised patches of rat mesenteric artery, inhibition of Ca²⁺ channels required millimolar concentrations of cytoplasmic Ca²⁺, suggesting a lower Ca²⁺ sensitivity of the channel (Huang et al., 1989). The reason for this discrepancy is unclear.

In the present study, a Hill coefficient close to 1 was calculated for the inhibition observed in cell-free patches, indicating interaction of Ca²⁺ with a single target site. This finding is in line with the idea that inhibition in excised patches is due to a direct interaction of Ca²⁺ with a binding site on the α1 subunit of the channel, which is a single EF-hand motif present both in cardiac and in smooth muscle channels (De Leon et al., 1995). In intact cells, accurate determination of the actual intracellular concentration of Ca²⁺, in particular at the cell membrane, is difficult. We have therefore employed two independent methods to estimate the level of intracellular free Ca²⁺; i.e., fura-2 fluorescence and open probability of Ca²⁺-activated K⁺ channels. Both methods yielded consistent results. When intracellular Ca²⁺ was elevated by exposure of the cells to A23187 plus 100 μM extracellular Ca²⁺, an intracellular Ca²⁺ concentration of ∼300 nM was obtained and Ca²⁺ channel activity was significantly suppressed. This result is in agreement with a previous study demonstrating that whole-cell Ba²⁺ currents in smooth muscle cells are inhibited when the cells' cytoplasmic Ca²⁺ concentration is elevated in the high nanomolar range (Ohya et al., 1988). Similarly, in cardiac muscle, a mechanism of Ca²⁺-dependent inhibition of L-type channels was detected that apparently required a rather modest rise in intracellular Ca²⁺; i.e., to levels below 1 μM (Ohya et al., 1988). These reports on inhibition of L-type channels in intact cells at submicromolar concentrations of cytoplasmic Ca²⁺ are in clear contrast to the observation that Ca²⁺-dependent inhibition of Ca²⁺ channels in excised patches was found to require concentrations above 1 μM (Romanin et al., 1992; Schmid et al., 1995). Thus, in intact cells, Ca²⁺-dependent inhibition occurred at about one order of magnitude lower concentrations with a substantially steeper (n_H ≅ 4) concentration dependence than in excised patches. These data suggest that elevation of bulk cytoplasmic Ca²⁺ up to the low micromolar range is able to suppress Ca²⁺ entry through L-type Ca²⁺ channels in intact smooth muscle cells via a rather complex cellular mechanism, while local elevation of Ca²⁺ at the cytoplasmic face of the channel above 1 μM induces inactivation via interaction with a single target site.

Role of Protein Phosphatase Type 2B

In an attempt to identify the Ca²⁺-dependent mechanism that suppresses Ca²⁺ channel activity in intact cells, we tested the involvement of putative indirect pathways. Up to now, several Ca²⁺-dependent signaling pathways have been implicated in the control of Ca²⁺ channel function. Our measurements of intracellular pH demonstrated that pH remained constant during the observed Ca²⁺-dependent suppression of channel activity.

L-type channel function is known to be governed via Ca²⁺-dependent protein kinase activity (Schuhmann and Groschner, 1994) as well as by Ca²⁺-dependent changes in the cells cytoskeleton (  Johnson and Byerly, 1994). Thus, we tested the kinase inhibitor H-7 and the cytoskeleton destabilizer cytochalasin B for its ability to interfere with Ca²⁺-dependent channel inhibition. Neither agent affected Ca²⁺-induced inhibition of L-type Ca²⁺ channel activity, arguing against a role of kinases and/or the cells cytoskeleton.

Since it has been proposed previously that functionality of Ca²⁺ channels requires basal phosphorylation of channel proteins, and that Ca²⁺-dependent dephosphorylation represents an important mechanism of downregulation of cellular Ca²⁺ channel activity (Chad and Eckert, 1986; Armstrong, 1989), we tested cyclosporin A, an inhibitor of the Ca²⁺-dependent phosphatase type 2B (calcineurin). Cyclosporin A by itself did not affect basal channel function, indicating that PP2B-dependent dephosphorylation is not involved in the control of Ca²⁺ channel function at basal, resting conditions. This is not surprising since PP2B is not expected to be active at the low level of intracellular free Ca²⁺ (50 nM) measured in our cell preparation under control conditions (Klee et al., 1979). Nonetheless, Ca²⁺-dependent inhibition of channel activity was completely occluded in cells pretreated with a relatively low concentration (1 μg/ml) of cylosporin A. Based on the known potency and specificity of cyclosporin A as an inhibitor of PP2B, this result suggests that PP2B may be involved in Ca²⁺-mediated negative feedback control of L-type Ca²⁺ channels in smooth muscle. Consequently, we tested whether PP2B is able to inhibit Ca²⁺ channel activity and to promote Ca²⁺-dependent inhibition of smooth muscle Ca²⁺ channels. Addition of purified PP2B in the presence of 1 μM calmodulin and 1 μM free Ca²⁺ resulted in a profound suppression of channel activity. Addition of calmodulin (1 μM) to the cytoplasmic side of excised patches failed to inhibit channel activity at 1 μM free Ca²⁺ in the absence of exogenous PP2B, arguing against the presence of endogenous PP2B in the patches of membrane. In the presence of PP2B, Ca²⁺-dependent inhibition of Ca²⁺ channel activity in excised patches exhibited a concentration dependence corresponding to that of Ca²⁺-dependent stimulation of PP2B phosphatase activity, indicating that the effects of PP2B are due to downregulation of Ca²⁺ channels by a Ca²⁺-dependent dephosphorylation process. The characteristics of this concentration dependence (i.e., IC₅₀ value and Hill coefficient) resembled those observed for Ca²⁺-dependent inhibition of Ca²⁺ channels in intact smooth muscle cells, supporting the idea that PP2B is involved in Ca²⁺-dependent control of L-type Ca²⁺ channel function in smooth muscle.

Modulation of Single Channel Properties by Cytoplasmic Ca²⁺ and PP2B

Analysis of the effects of cytoplasmic Ca²⁺ on single channel properties revealed a striking difference between Ca²⁺-dependent modulation of channels in intact cells and in cell-free patches. In contrast to the inhibition of channels in inside-out patches, which was mainly due to a reduction in P_o, inhibition of Ca²⁺ channels in intact cells was based on a reduction of both P_o and P_s. The observation of a Ca²⁺-induced low P_o gating mode in cell-free membranes is consistent with the recently proposed model of direct binding of Ca²⁺ ions to an EF-hand motif of the channels' α1 subunit, which results in typically low P_o gating (“mode Ca”) of the channel (Imredy and Yue, 1994; De Leon et al., 1995). Nonetheless, a specific effect of cytoplasmic Ca²⁺ on channels in intact smooth muscle cells was reduction of P_s. This reduction of P_s was not induced by elevation of Ca²⁺ at the cytoplasmic side of cell-free patches, but mimicked when Ca²⁺ was elevated in the low micromolar range in the presence of PP2B. These results of the present study demonstrate for the first time Ca²⁺- induced suppression of the availability of Ca²⁺ channels in intact smooth muscle cells and in cell-free membranes exposed to PP2B. We have recently reported on the downregulation of smooth muscle L-type Ca²⁺ channels by PP2A-induced dephosphorylation (Groschner et al., 1996). In contrast to channel modulation by PP2B, PP2A did not suppress channel availability but affected specifically fast gating properties (P_o) of the channels by suppression of long lasting channel openings (mode 2 gating). Interestingly, the reduction of P_o observed in the presence of PP2B was not based on suppression of mode 2 gating, as evident from two experiments that allowed analysis of open time distributions (our unpublished observations). Taken together, these data strongly support the idea of multiple regulatory phosphorylation sites that are dephosphorylated in a rather selective manner by specific phosphatases and control different kinetic properties of the channel.

Physiological and Pharmacological Implications

Fine adjustment of Ca²⁺ entry according to the actual Ca²⁺ concentration in the cytosol is an important cellular mechanism. The results of the present study suggest that in vascular smooth muscle, this negative feedback control involves at least two sensors for Ca²⁺ within the cell. One of these sensors appears to be located close to the Ca²⁺ entry pathway, while another more remote sensor detects changes in the bulk cytosolic Ca²⁺ concentration. Albeit the present study does not provide information as to how Ca²⁺ affects channel function by its permeation through the channel pore, it is clearly demonstrated that the cytoplasmic Ca²⁺ controls channel function through two distinct intracellular mechanisms. The existence of multiple feedback mechanisms is not unexpected since cellular Ca²⁺ homeostasis is based on a variety of mechanisms, including Ca²⁺ transport across the plasma membrane and across the membrane of intracellular Ca²⁺ stores. It is already well established that Ca²⁺ signaling within a cell involves Ca²⁺ gradients (van Breemen et al., 1995). According to the present results, elevation of intracellular Ca²⁺ within the smooth muscle cell causes a reduction of Ca²⁺ entry due to PP2B-mediated dephosphorylation that may take place even at low or moderate rates of Ca²⁺ entry. On the other hand, the rate of Ca²⁺ entry through the L-type channel is unequivocally subject to a tight, local feedback mechanism. This local mechanism is likely to serve rapid, short term modulation of Ca²⁺ entry. Nonetheless, both the local and the remote mechanism may be of particular importance for smooth muscle Ca²⁺ homeostasis. Suppression of one of these mechanisms may produce severe disturbances in smooth muscle Ca²⁺ homeostasis. In the present study, we demonstrate that cyclosporin A, which is widely used as an immunosuppressant drug, effectively inhibits the remote Ca²⁺-dependent control of L-type Ca²⁺ channels in human vascular smooth muscle. The interference of cyclosporin A with the negative feedback control of Ca²⁺ entry may contribute significantly to the severe vascular side effects (Sturrock et al., 1992) (i.e., promotion of vasoconstriction and hypertension) observed during cyclosporin A treatment.

In summary, our results demonstrate that in smooth muscle cells, the cytoplasmic Ca²⁺ concentration governs the function of L-type Ca²⁺ channels via two distinct negative feedback systems involving both Ca²⁺- dependent dephosphorylation and direct Ca²⁺ binding.