Extracellular Ba2‡ and voltage interact in stomatal guard cells

 

Extracellular Ba2‡ and voltage interact to gate Ca2‡ channels at the plasma membrane of stomatal guard cells

by Hamilton D. W. A.,  Hills A., Blatt M. R. (2001)

in FEBS Letters 491 (2001) 99-103 – 

http://onlinelibrary.wiley.com/store/10.1016/S0014-5793(01)02176-7/asset/feb2s0014579301021767.pdf?v=1&t=ip5xzwp2&s=16a1dd5e6e60dc5a7e36dea9e88360ec36821ea3

Abstract

Ca2+ channels at the plasma membrane of stomatal guard cells contribute to increases in cytosolic free [Ca2+] ([Ca2+]i) that regulate K+ and Cl3 channels for stomatal closure in higher-plant leaves.

Under voltage clamp, the initial rate of increase in [Ca2+]i in guard cells is sensitive to the extracellular divalent concentration, suggesting a close interaction between the permeant ion and channel gating.

To test this idea, we recorded single-channel currents across the Vicia guard cell plasma membrane using Ba2+ as a charge carrying ion. Unlike other Ca2+ channels characterised to date, these channels activate at hyperpolarising voltages.

We found that the open probability (Po) increased strongly with external Ba2+ concentration, consistent with a 4-fold cooperative action of Ba2+ in which its binding promoted channel opening in the steady state.

Dwell time analyses indicated the presence of a single open state and at least three closed states of the channel, and showed that both hyperpolarising voltage and external Ba2+ concentration prolonged channel residence in the open state.

Remarkably, increasing Ba2+ concentration also enhanced the sensitivity of the open channel to membrane voltage.

We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the voltage gate.

ABA, Ca2+ and stomatal guard cells

Photo credit: NCBI

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.

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

by Hamilton D. W. A.,  Hills A., Kohler B., Blatt M. R. (2000)

in Proc. Natl Acad. Sci. USA, 97, 4967–4972. –

CrossRef | PubMed | CAS | ADS

http://www.ncbi.nlm.nih.gov/pubmed/10781106

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.