MODULATION OF K+ CHANNELS IN VICIA STOMATAL GUARD-CELLS BY PEPTIDE HOMOLOGS TO THE AUXIN-BINDING PROTEIN-C TERMINUS

被引:130
作者
THIEL, G
BLATT, MR
FRICKER, MD
WHITE, IR
MILLNER, P
机构
[1] UNIV GOTTINGEN, INST PFLANZENPHYSIOL, W-3400 GOTTINGEN, GERMANY
[2] UNIV OXFORD, DEPT PLANT SCI, OXFORD OX1 3RB, ENGLAND
[3] UNIV LEEDS, DEPT BIOCHEM & MOLEC BIOL, LEEDS LS2 9JT, W YORKSHIRE, ENGLAND
关键词
SYNTHETIC OLIGOPEPTIDE; AUXIN SIGNAL TRANSDUCTION; LASER-SCANNING CONFOCAL MICROSCOPY; CYTOPLASMIC PH;
D O I
10.1073/pnas.90.24.11493
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Transduction of the auxin stimulus in plants is thought to entail binding of the hormone to a soluble auxin-binding protein (ABP) outside the cell and subsequent interaction between this auxin-protein complex and an integral membrane receptor (''docking'') protein that couples the signal across the plasma membrane. To explore the structural requirements for ABP function, synthetic peptides were prepared to the amino acid sequences of the predicted surface domains of ABPzm1, the dominant ABP from Zea. Biological function was assayed under voltage clamp, monitoring the ability of the peptides to evoke auxin-related modulations in inward- (I(K,in)) and outward-rectifying (I(K,out)) K+ channel activities of Vicia guard cells in the absence of added auxin. Only the peptide corresponding to the C-terminal domain of ABPzm1 was active. The dominant response was an inactivation of I(K,in), although the peptide also evoked an activation of I(K,out). Inactivation of I(K,in) was complete within 20-30 s and was fully reversible, was marked by a slowing of voltage-dependent activation and deactivation, and was dependent on peptide concentration (K1/2, 16 +/- 6 muM). Buffering cytoplasmic-free [Ca2+] with EGTA had no effect on I(K,in) response to the peptide. However, virtually complete and reversible block of the response was achieved when cytoplasmic pH (pH(i)) was brought under experimental control using the weak acid butyrate. Parallel measurements of pH(i) using the fluorescent dye 2',7'-bis(2-carboxyethyl-5(6)-carboxyfluorescein (BCECF) and dual-wavelength laser-scanning confocal microscopy demonstrated that the C-terminal peptide evoked rapid and reversible cytoplasmic alkalinizations of 0.4 +/- 0.1 pH(i) unit and confirmed the antagonism of the pH(i) response in the presence of butyrate. These, and comparable results with the auxins indole acetic acid and 1-naphthyleneacetic acid, implicate the C-terminal domain of ABPzm1 in auxin-ABP coupling to pH(i) and an associated intracellular signaling cascade.
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页码:11493 / 11497
页数:5
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