Mechanism of the Inhibition of Ca2+-Activated Cl− Currents by Phosphorylation in Pulmonary Arterial Smooth Muscle Cells

The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl− currents (IClCa) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, IClCa was evoked immediately upon membrane rupture but then exhibited marked rundown to ∼20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5′-(β,γ-imido)-triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, IClCa was ∼100% of initial levels. IClCa recorded with AMP-PNP–containing pipette solutions were significantly larger than control currents and had faster kinetics at positive potentials and slower deactivation kinetics at negative potentials. The marked increase in IClCa was due to a negative shift in the voltage dependence of activation and not due to an increase in the apparent binding affinity for Ca2+. Mathematical simulations were carried out based on gating schemes involving voltage-independent binding of three Ca2+, each binding step resulting in channel opening at fixed calcium but progressively greater “on” rates, and voltage-dependent closing steps (“off” rates). Our model reproduced well the Ca2+ and voltage dependence of IClCa as well as its kinetic properties. The impact of global phosphorylation could be well mimicked by alterations in the magnitude, voltage dependence, and state of the gating variable of the channel closure rates. These data reveal that the phosphorylation status of the Ca2+-activated Cl− channel complex influences current generation dramatically through one or more critical voltage-dependent steps.


D E T A I L E D K I N E T I C A N A Ly S I S o f I C L C A R E C o R D E D I N M y o C y T E S D I A Ly z E D w I T h A T P o R A M P -P N P
Fig. S1 A shows graphs reporting the Ca 2+ sensitivity of the time constant of activation (τ act ) of I ClCa with ATP (filled squares) or AMP-PNP (empty squares) at +70 mV (a), +90 mV (b), +110 mV (c), and +130 mV (d). In the presence of ATP, τ act was Ca 2+ independent at the voltages examined (slopes were not significantly different from 0 with P values ranging from 0.066 to 0.389). In contrast with AMP-PNP, τ act declined exponentially as a function of pipette [Ca 2+ ], and the values were significantly different from those obtained in ATP for Ca 2+ concentrations > 250 nM. Fig. S1 B shows graphs reporting the impact of ATP and AMP-PNP on the Ca 2+ dependence of the time constant(s) of deactivation (τ deact ) at −100 mV (a), −50 mV (b), −10 mV (c), and +50 mV (d). Although deactivation was mono-exponential for 250 and 500 nM Ca 2+ with both nucleotides, deactivation became bi-exponential with 1000 nM Ca 2+ and ATP, and with 750 nM and 1000 nM Ca 2+ in the presence of AMP-PNP. For both nucleotides, the slow component of deactivation (τ slow ) increased exponentially as a function of pipette [Ca 2+ ] in the negative range of membrane potentials (compare panels Ba, b, and c with panel Bd; the fast component of deactivation τ fast was not plotted for clarity). We also analyzed the voltage dependence of the ratio of the amplitude of the slow over fast (Amp slow/fast ) components of deactivation with 1000 nM Ca 2+ . Although this parameter was voltage insensitive between −100 mV and −20 mV for both nucleotides, the analysis revealed a significant increase of this parameter with AMP-PNP vs. ATP (two-way ANOVA yielded P < 0.001; unpublished data); e.g., at −100 mV, Amp slow/fast was 0.33 ± 0.06 and 0.60 ± 0.06 (P < 0.001) with ATP and AMP-PNP, respectively. Thus, general dephosphorylation slowed deactivation kinetics, especially at higher levels of intracellular Ca 2+ levels and negative potentials; at 1000 nM Ca 2+ , in addition to slowing deactivation at negative potentials, dephosphorylation increased the proportion of channels entering slow deactivation in the negative range of membrane potentials.
We also investigated the effects of AMP-PNP on the voltage dependence of I ClCa kinetics and the data are summarized in Fig. S2. Except for ATP and 250 nM Ca 2+ , where regression analysis revealed a slope significantly different from 0 (P = 0.035), τ act did not vary as a function of voltage in cells dialyzed with ATP or AMP-PNP at all pipette [Ca 2+ ] tested (P ≥ 0.298; Fig. S2 Aa−d). However, τ act was significantly reduced in AMP-PNP-containing myocytes dialyzed with 500 nM Ca 2+ or higher, and this effect was proportionately greater as pipette [Ca 2+ ] increased. The time constant of deactivation of I ClCa (τ deact ) decreased with membrane hyperpolarization in cells dialyzed with ATP and pipette [Ca 2+ ] in the range of 250-750 nM Ca 2+ (Fig. S2 Ba−c), a result consistent with a previous report from our group in rabbit pulmonary artery myocytes (Greenwood et al., 2001). In contrast, neither the fast (labeled as τ fast ) nor the slow decay (labeled as τ slow ) of currents evoked by 1000 nM Ca 2+ exhibited any voltage dependence ( . τ slow evoked by 750 and 1000 nM Ca 2+ was also slower in the presence of AMP-PNP, whereas τ fast was slightly accelerated (Fig. S2 Bd). Although the appearance of τ fast at higher pipette [Ca 2+ ] is unclear, our results indicate that global dephosphorylation in the absence of ATP tends to lock the channel in the open state at negative membrane potentials irrespective of the Ca 2+ concentration, which is consistent with the observed negative shift in the voltage dependence of I ClCa by AMP-PNP.
Our computer simulations to mimic the behavior of I ClCa in pulmonary arterial myocytes dialyzed with ATP and AMP-PNP were conducted using two mathematical models, each run with their own set of parameters (Table I) to generate the data presented in Figs. 8 and 9. As in our experiments, all simulations were performed in the absence of a Cl − gradient across the membrane (E Cl = 0 mV). Because the affinity of I ClCa for Ca 2+ was found to be four to five times higher in pulmonary artery myocytes than frog oocytes (Kuruma and Hartzell, 2000), the binding rate constant k on was increased to 20 M −1 s −1 instead of 3 M −1 s −1 , while a value (50 s −1 ) identical to that of Kuruma and Hartzell (2000) was used as the unbinding rate constant k off . Although the values used for the rate of channel opening were different, they followed a similar trend to those used by Kuruma and Hartzell (2000), increasing with the number of Ca 2+ ions bound (Table I). Another distinction from the model used in frog oocytes is that the equations describing the voltage dependence of the rate of channel closure were Boltzmann relationships instead of exponen-tial functions. This was necessary to describe the sigmoidal nature of the voltage dependence of the Ca 2+ affinity curves determined in our experiments (Fig. 5 A). The voltage dependence of the function determining β x was progressively made more negative with Ca 2+ binding (Table I) to account for the negative shift in the voltage dependence of I ClCa conductance (Fig. 6 A). The steepness of the Boltzmann relationships describing β x was set to a constant value of 50 mV (Table I) for both models and is similar to the range of values estimated from experiments ( Fig. 6 A,  inset). The gating variable of the O 2 and O 3 states for the ATP model was set to 0 instead of 1 in the AMP-PNP model. As described in the next supplementary section, this procedure greatly attenuated the steep Ca 2+ dependence of the time constant of activation of I ClCa observed with AMP-PNP, which was Ca 2+ independent in cells dialyzed with ATP (Fig. S1). Setting these states to 0 allowed for locking a fraction of the channels in the "closed" or "blocked" state at [Ca 2+ ] i , thus opposing the activating effect of Ca 2+ on I ClCa kinetics, as described in the following section.

C o M P A R I S o N o f K I N E T I C S o f I C L C A A N A Ly z E D f R o M E x P E R I M E N T A L A N D M o D E L E D D A T A
As mentioned in the text, our models reproduced well the Ca 2+ and voltage dependence of I ClCa and how these properties are modulated by global phosphorylation status. We also performed a superficial comparison of kinetic data derived from actual experiments and our computer models. For example with 500 nM Ca 2+ , τ act at +120 mV was 216 ms (actual data: 432 ± 28 ms, n = 7) and 193 ms (actual data: 163 ± 27 ms, n = 7), respectively, with ATP and AMP-PNP; τ deact at −50 mV was 54 ms (actual data: 105 ± 7 ms, n = 6) with ATP and 139 ms for the slow component figure S1. Ca 2+ dependence of I ClCa kinetics analyzed after 20 min of cell dialysis with ATP and AMP-PNP. (A) Panels a-d show graphs reporting mean ± SEM time constant of activation (τ act ) measured after 20 min of cell dialysis with 3 mM ATP (filled squares; n = 5-9) or 3 mM AMP-PNP (empty squares; n = 4-8) as function of internal Ca 2+ concentration ([Ca 2+ ] i ) at the four distinct step potentials indicated: +70 mV (a), +90 mV (b), +110 mV (c), and +130 mV (d). Time constants were determined by exponential curve fitting of the time-dependent component of I ClCa elicited during 1-s depolarizing steps from HP = −50 mV (see Fig. 3). The straight lines passing through all datasets obtained in ATP are linear regression fits whose slopes were all not significantly different from 0 (P > 0.05), indicating lack of Ca 2+ dependence. All lines passing through the AMP-PNP datasets are B-Spline fits estimated by Origin software. Two-way ANOVA tests revealed significant differences at all potentials examined between ATP and AMP-PNP (P < 0.01); post-hoc tests: † , P < 0.01; ‡ , P < 0.001. (B) Panels a-d show plots reporting mean ± SEM time constant of deactivation (τ deact ) measured after 20 min of cell dialysis with 3 mM ATP (filled squares; n = 3-7) or 3 mM AMP-PNP (empty squares; n = 2-9) as function of internal pipette Ca 2+ concentration ([Ca 2+ ] i ) at the four representative step potentials indicated: −100 mV (a), −50 mV (b), −10 mV (c), and +50 mV (d). Time constants were determined by exponential curve fitting of I ClCa tail currents. The double-pulse protocol used to elicit tail currents consisted of a constant 1-s step to +100 mV followed by a 1-s return step ranging from −100 to +50 mV applied in 10-mV increments from HP = −50 mV. As explained in the text, deactivation was bi-exponential with ATP and 1000 nM Ca 2+ , and with AMP-PNP and 750 nM and 1000 nM Ca 2+ . Only the slow time constant of deactivation is reported in this graph for the sake of clarity. The lines passing through all datasets are B-Spline fits estimated by Origin software. Two-way ANOVA tests revealed significant differences at all potentials examined between ATP and AMP-PNP (P < 0.01) except +50 mV (n.s., not significant); posthoc tests: *, P < 0.05; † , P < 0.01; ‡ , P < 0.001. All plots were generated from data obtained after 20 min of cell dialysis with either nucleotide.
figure S2. Voltage dependence of I ClCa kinetics analyzed after 20 min of cell dialysis with ATP and AMP-PNP. (A) Panels a-d show graphs reporting mean ± SEM time constant of activation (τ act ) measured after 20 min of cell dialysis with 3 mM ATP (filled squares; n = 4-8) or 3 mM AMP-PNP (empty squares; n = 4-8) as function of step potential at the four Ca 2+ concentrations indicated: 250 nM (a), 500 nM (b), 750 nM (c), and 1000 nM (d). Time constants were determined by exponential curve fitting of the time-dependent component of I ClCa elicited during 1-s depolarizing steps from HP = −50 mV (see Fig.  3). All straight lines through the data points are linear regressions. Except for 250 nM Ca 2+ , two-way ANOVA analyses revealed significant differences between ATP and AMP-PNP datasets (P < 0.001); post-hoc tests: † , P < 0.01; ‡ , P < 0.001. (B) Panels a-d show plots reporting mean ± SEM time constant(s) of deactivation (τ deact ) measured after 20 min of cell dialysis with 3 mM ATP (filled symbols; n = 2-8) or 3 mM AMP-PNP (empty symbols; n = 2-9) as function of step potential at the four Ca 2+ concentrations indicated: 250 nM (a), 500 nM (b), 750 nM (c), and 1000 nM (d). Time constants were determined by exponential curve fitting of I ClCa tail currents. The double-pulse protocol used to elicit tail currents consisted of a constant 1-s step to +100 mV followed by a 1-s return step ranging from −100 to +50 mV applied in 10-mV increments from HP = −50 mV. As explained in the text, deactivation was bi-exponential with ATP and 1000 nM Ca 2+ (n = 5-7), and with AMP-PNP and 750 nM (n = 2-5) and 1000 nM Ca 2+ (n = 2-9). Fast (τ fast ) and slow (τ slow ) time constants of deactivation are labeled as circles and squares, respectively. Except for the data with ATP and 250 nM and 500 nM Ca 2+ , and with AMP-PNP and 500 nM Ca 2+ , which are exponential fits to the data, all other lines are linear regressions. For all groups of data, two-way ANOVA analyses revealed significant differences between ATP and AMP-PNP with P < 0.05; post-hoc tests: † , P < 0.01; ‡ , P < 0.001. Some, but not all, mean data points presented here are reproduced from Fig. 8 but plotted differently. All plots were generated from data obtained after 20 min of cell dialysis with either nucleotide.