Abstract

Acid-induced potassium uptake through K+ channels is a prerequisite for stomatal opening. Our previous studies identified a pore histidine as a major component of the acid activation mechanism of the potato guard cell K+ channel KST1 (1). Although this histidine is highly conserved among all plant K+ uptake channels cloned so far, the pH-dependent gating of the Arabidopsis thaliana guard cell K+ channel KAT1 was not affected by mutations of this histidine. In both channels, KST1 and KAT1, aspartate mutants in the K+ channel consensus sequence GYGD adjacent to the histidine (KST1-D269N and KAT1-D265N) were inhibited by a rise in the extracellular proton concentration. pH changes affected the half-maximal activation voltage V(1)/(2) of the KST1 mutant, whereas in the mutant channel KAT1-D265N an acid-induced decrease in the maximum conductance gmax indicated the presence of a proton block. In contrast to the wild type KST1, the S4-mutant channel KST1-R181Q exhibited an activation upon alcalization of the extracellular solution. From our electrophysiological studies on channel mutants with respect to the pore histidine as well as the aspartate, we conclude that the common proton-supported shift in the voltage dependence of KST1 and KAT1 is based on distinct molecular elements.

Highlights

  • In the stomatal complex, acid-activated Kϩ uptake into guard cells through inward rectifying Kϩ channels (Kiϩn channels) is fundamental for the turgor-driven volume changes in guard cells [2,3,4,5]

  • The Role of Histidines for pH Sensing in KAT1—To prove the proposed plant-specific mechanism for acid activation in Kϩ uptake channels [1], the pH dependence of KAT1 wild type and channel mutants with respect to the conserved pore histidine was studied after expression in Xenopus oocytes

  • Because additional histidines were not located on the putative extracellular face of the membrane in KAT1, the tested pH range was extended to pH 4.5 to screen for more acidic residues like aspartate and glutamate, which could account for acid activation in KAT1

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Summary

EXPERIMENTAL PROCEDURES

Generation of Channel Mutants—KST1 and KAT1 single histidine and glutamate mutants were generated as described previously [1, 24]. For the generation of aspartate mutants, site-directed mutagenesis (QuikChangeTM Site-directed Mutagenesis kit, Stratagene, Heidelberg, Germany) with primers 5Ј-ACCGGTTATGGAAACTTGCATGCTGAG-3Ј (KST1-D269N) and 5Ј-CCACGGGATATGGAAATTTTCATGCTGAGAACCC-3Ј (KAT1-D265N) was performed on plasmids pKST1#8 and pKAT1 in the pGEMHE vector [25]. Electrophysiology—cRNAs of wild type and mutant channels were generated by in vitro transcription (T7-Megascript kit, Ambion Inc.) and injected in oocytes of Xenopus laevis (Nasco, Fort Atkinson, WI) using a General Valve Picospritzer II microinjector (Fairfield, NJ). PH-dependent currents of the aspartate mutants were recorded in the absence of Ca2ϩ. TEAϩ blocking experiments were performed at pH 5.6, and TEAϩ was added to a final concentration of 0.1, 0.5, 1, 5, 10, and 20 mM, respectively. All solutions were adjusted to a final osmolality of 215–235 mosmol/kg with D-sorbitol. Following activation pulses of 1.5–5 s in duration in the range of ϩ20 mV to Ϫ150 mV, inward currents relaxed during the second pulse to Ϫ70 mV

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RESULTS
DISCUSSION
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