Ca2+ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and abscisic acid

Abstract

In stomatal guard cells of higher-plant leaves, abscisic acid (ABA) evokes increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) by means of Ca(2+) entry from outside and release from intracellular stores. The mechanism(s) for Ca(2+) flux across the plasma membrane is poorly understood. Because [Ca(2+)](i) increases are voltage-sensitive, we suspected a Ca(2+) channel at the guard cell plasma membrane that activates on hyperpolarization and is regulated by ABA. We recorded single-channel currents across the Vicia guard cell plasma membrane using Ba(2+) as a charge-carrying ion. Both cell-attached and excised-patch measurements uncovered single-channel events with a maximum conductance of 12.8 +/- 0.4 pS and a high selectivity for Ba(2+) (and Ca(2+)) over K(+) and Cl(-). Unlike other Ca(2+) channels characterized to date, these channels rectified strongly toward negative voltages with an open probability (P(o)) that increased with [Ba(2+)] outside and decreased roughly 10-fold when [Ca(2+)](i) was raised from 200 nM to 2 microM. Adding 20 microM ABA increased P(o), initially by 63- to 260-fold; in both cell-attached and excised patches, it shifted the voltage sensitivity for channel activation, and evoked damped oscillations in P(o) with periods near 50 s. A similar, but delayed response was observed in 0.1 microM ABA. These results identify a Ca(2+)-selective channel that can account for Ca(2+) influx and increases in [Ca(2+)](i) triggered by voltage and ABA, and they imply a close physical coupling at the plasma membrane between ABA perception and Ca(2+) channel control.

Figure 1

Ca²⁺ channels at the plasma membrane of Vicia guard cells uncovered at negative voltages. (A) Representative whole-cell record with voltage clamped to a 30-s ramp from +50 to −200 mV with 30 mM Ba²⁺ in bath and 30 mM Ba²⁺ in the pipette. (Inset) Cell-attached records with voltages (in mV) calculated assuming that a protoplast voltage of −50 mV, close to the voltage obtained in whole-cell recordings with the current clamped to zero. (B) Current from one outside-out patch with 30 mM Ba²⁺ in the pipette (inside) and 10 mM Ba²⁺ in the bath. Voltages (Left) in mV. (Insets, Right) Point-amplitude histograms (27) used to estimate open-channel amplitudes (see Methods; note the log scale) and single-channel events at −150 mV shown on an expanded time scale. (C) Current from the same patch as in B after adding 100 μM LaCl₃ to the bath. Scale (A–C): vertical, 2 pA (A, Inset: 4 pA); horizontal, 1 s (A, Inset: 2 s; B, Inset: 150 ms). (D) Means ± SEM of open-channel currents from outside-out patches (n = 9) with 30 mM Ba²⁺ inside and 2 (■), 10 (▴), and 30 mM Ba²⁺ (●) outside. Currents from cell-attached patches (○, n = 8) as in A but with voltage uncorrected for comparison of γ_Ba. Regression analyses (lines) of these data extrapolated to the voltage axis to determine E_rev. Means ± SEM of currents from separate measurements with addition of 20 mM Cl⁻ as BaCl₂ (▵, n = 5) and with 10 mM Ca²⁺ (◊, n = 4) in place of Ba²⁺ outside included for comparison. (Inset) γ_Ba plotted against [Ba²⁺] and fitted to a simple hyperbolic function.

Figure 2

Ca²⁺ channel open probability (P_o) is elevated by membrane hyperpolarization and [Ba²⁺] outside. Means ± SE of P_o from mean open times of 100-s recordings including data of Fig. 1 with 2 (■), 10 (▴), and 30 mM Ba²⁺ (●) outside, and with 10 mM Ca²⁺ (◊) in place of Ba²⁺ outside. Mean ± SE of measurements at −100 mV (n = 3) with 10 mM Ba²⁺ inside and 30 mM Ba²⁺ outside (○, compare ●) show the effect of changing [Ba²⁺] inside. Curves are empirical fittings to a common exponential function.

Figure 3

P_o is suppressed by micromolar [Ca²⁺]_i. Means ± SE of P_o from mean open times of 100-s recordings at −120 mV (n = 3). Ca²⁺ added on the cytosolic side (inside) during inside-out recordings against a background of 30 mM Ba²⁺ and with 10 mM Ba²⁺ outside. (Insets) Segments of traces at each [Ca²⁺]_i. Data from one patch. Scale: vertical, 1 pA; horizontal, 1 s.

Figure 4

ABA activates Ca²⁺ channels. (A) Records from two patches, cell-attached (Left) and excised inside-out (Right) before (Top) and after (Bottom) adding 20 μM ABA to the bath. Traces in ABA at 55 s (cell-attached) and 20 s (excised patch) after additions. Membrane voltage clamped to −100 mV and with 30 mM Ba²⁺ in bath and pipette (cell-attached) or with 30 mM Ba²⁺ inside and 10 mM Ba²⁺ outside (excised patch). Scale: vertical, 2 pA; horizontal, 1 s. Point-amplitude histograms of channel openings for excised patch data plotted on a log scale (far right): events −ABA taken over 100 s; events +ABA taken over 5 s and scaled by a factor of 20 for comparison. (B) Current–voltage curves obtained by summing currents recorded from one excised, inside-out patch during 10-s voltage ramps between 0 and −200 mV. Baseline (leakage) current corrected by subtracting corresponding data (−ABA) with no opening events. Data are the sum of 40 ramps (−ABA) and 10 ramps (+ABA) taken immediately before and after adding ABA. Sum before ABA addition scaled by 0.25 for comparison, and both current-voltage curves fitted to a Boltzmann function of the form 1 where g_max is the relative conductance maximum, V the clamp voltage, E_Ba the Ba²⁺ equilibrium voltage (= −14 mV), δ the voltage sensitivity factor, V_1/2 the voltage giving half-maximal current activation, and z, F, R, and T have their usual meanings. Fitted parameters (+ABA): δ, 0.93 ± 0.05; V_1/2, −83 ± 2 mV. Fittings of currents summed before adding ABA gave V_1/2, −142 ± 48 mV if δ was constrained to a value of unity. Variation in P_o (see Fig. 5) accounts for the lower activation by ABA when summed over 50 s.

Figure 5

ABA, but not voltage steps, evokes oscillations in Ca²⁺ channel activity. P_o (data points) and relative net charge, Q (solid lines), calculated at consecutive 5-s intervals in six independent experiments (a–f, three cell-attached, three excised, inside-out patches). Note the different P_o scales (Right) for each experiment. Q calculated by summing all points above the zero current level during each 5-s interval and scaled to P_o ranges. Segments of current traces c and d (including data from Fig. 4) with closed level (−c) as indicated. Scale: vertical, 2 pA; horizontal, 1 s. Times of ABA addition indicated (|, Left) in each experiment and aligned between c and e. Voltage stepped from −100 to −150 mV at 50 s in f.