The molecular mechanisms by which higher plant cells take up K' across the plasma membrane (plasma- Inward-rectifying K+ channels (IK+,in channels) were identified in guard cells (11,12) and have been found in several higher plant cell types, including root cells (refs. 13-17; for a review, see ref. 18). These IK+,in channels have been suggested to provide a major mechanism for K+ uptake across the plasma membrane of guard cells during stomatal opening. IK+,in channels are activated by membrane potentials more negative than approximately -90 mV and allow selective K+ influx into higher plant cells (11,13,16,19). Light-activated electrogenic proton-extruding pumps, which hyperpolarize the plasma membrane (19-23), provide the electrochemical driving force for IK+ in channel-mediated KV uptake. Support for the hypothesis that IK+,i channels constitute a major pathway for KV uptake during stomatal opening has been gained from detailed studies on Viciafaba guard cells, which resulted in the following findings. (i) Average physiological K+-uptake currents during stomatal opening of -10 pA per guard cell (24) can be carried by IK+ in channels (11,12). (ii) The K+ selectivity with respect to other alkali metal ions and steady-state activation properties of IK+,in channels support physiological properties of the ionic specificity and long-term K+ accumulation into guard cells (11,12,19). (iii) Aluminum ions, which inhibit stomatal opening (25), block IK+,in channels at similar concentrations (19). (iv) Elevation of the cytosolic Ca2+ concentration to micromolar levels inhibits IK+,in channels (26), which correlates to Ca2+ inhibition of stomatal opening and reduction of K+ uptake (27,28). In the present study the contribution of IK+,in channels to physiological K+ uptake fluxes was investigated by examining effects of naturally occurring changes in the extracellular K+ concentration (9, 24) on K+-channel properties and on electrical characteristics of the plasma membrane. lemma MATERIALS AND METHODS 11583The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Guard cells modulate stomatal apertures in response to hormones, metabolic demands and environmental stimuli. The guard cell PM H(+)-ATPases play a critical role in this process by generating the electrochemical gradient to drive solute transport and concomitant water flux. The PM H(+)-ATPase activity is specifically regulated by red and blue light, fungal toxins and auxin. To determine if the unique responsiveness of the guard cell PM H(+)-ATPase is due to the expression of a cell-specific isoform, we amplified by PCR, and cloned portions of PM H(+)-ATPase genes VHA1 and VHA2, which are expressed in guard cell protoplasts (GCP). In situ hybridization to leaf tissue sections indicated that VHA1 and VHA2 genes were expressed in guard cells and mesophyll cells but not in epidermal cells or vascular tissues. Furthermore, a gene-specific quantitative reverse transcription (RT)-PCR detected VHA1 and VHA2 mRNAs in both GCP and mesophyll cell protoplast mRNA as well as in mRNA isolated from roots, leaves, stems and flowers. Thus, two PM H(+)-ATPase genes expressed in guard cells are also expressed in many other tissues and cell types. This suggests that the unique responsiveness of the guard cell PM H(+)-ATPases to environmental stimuli results from cell-specific signal transduction pathways rather than the expression of a cell-specific PM H(+)-ATPase.

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labelling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. Cys530A1a mutant), C381 becomes the site of estrogen-compatible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed and region-specific chemical mutagenesis have identified a region around C530 important in discrimination between estrogens and antiestrogens, and other mutants have allowed identification of residues important in hormone-dependent transcriptional activation. Some transcriptionally inactive ER mutants also function as potent dominant negative ERs, suppressing the activity of wild-type ERs at low concentrations. These studies are beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer.

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