Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters.

A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.

Durotropic Growth of Pollen Tubes.

To reach the female gametophyte, growing pollen tubes must penetrate different tissues within the pistil, the female reproductive organ of a flower. Past research has identified various chemotropic cues that guide pollen tubes through the transmitting tract of the pistil, which represents the longest segment of its growth path. In addition, physical mechanisms also play a role in pollen tube guidance; however, these processes remain poorly understood. Here we show that pollen tubes from plants with solid transmitting tracts actively respond to the stiffness of the environment. We found that pollen tubes from Nicotiana tabacum and other plant species with a solid or semisolid transmitting tract increase their growth rate in response to an increasing matrix stiffness. By contrast, pollen tubes from Lilium longiflorum and other plant species with a hollow transmitting tract decrease their growth rate with increasing matrix stiffness, even though the forces needed to maintain a constant growth rate remain far below the maximum penetration force these pollen tubes are able to generate. Moreover, when confronted with a transition from a softer to a stiffer matrix, pollen tubes from N. tabacum display a greater ability to penetrate into a stiffer matrix compared with pollen tubes from L. longiflorum, even though the maximum force generated by pollen tubes from N. tabacum (11 µN) is smaller than the maximum force generated by pollen tubes from L. longiflorum (36 µN). These findings demonstrate a mechano-sensitive growth behavior, termed here durotropic growth, that is only expressed in pollen tubes from plants with a solid or semisolid transmitting tract and thus may contribute to an effective pollen tube guidance within the pistil.

Multiple cyclic nucleotide-gated channels coordinate calcium oscillations and polar growth of root hairs.

Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip-focused Ca2+ -gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide-gated channel family are required to maintain cytosolic Ca2+ oscillations and the normal growth of root hairs. CNGC6, CNGC9 and CNGC14 were expressed in root hairs, with CNGC9 displaying the highest root hair specificity. In individual channel mutants, morphological defects including root hair swelling and branching, as well as bursting, were observed. The developmental phenotypes were amplified in the three cngc double mutant combinations. Finally, cngc6/9/14 triple mutants only developed bulging trichoblasts and could not form normal root hair protrusions because they burst after the transition to the rapid growth phase. Prior to developmental defects, single and double mutants showed increasingly disturbed patterns of Ca2+ oscillations. We conclude that CNGC6, CNGC9 and CNGC14 fulfill partially but not fully redundant functions in generating and maintaining tip-focused Ca2+ oscillations, which are fundamental for proper root hair growth and polarity. Furthermore, the results suggest that these calmodulin-binding and Ca2+ -permeable channels organize a robust tip-focused oscillatory calcium gradient, which is not essential for root hair initiation but is required to control the integrity of the root hair after the transition to the rapid growth phase. Our findings also show that root hairs possess a large ability to compensate calcium-signaling defects, and add new players to the regulatory network, which coordinates cell wall properties and cell expansion during polar root hair growth.

Phosphatidylinositol-3,5-bisphosphate lipid-binding-induced activation of the human two-pore channel 2.

Mammalian two-pore channels (TPCs) are activated by the low-abundance membrane lipid phosphatidyl-(3,5)-bisphosphate (PI(3,5)P2) present in the endo-lysosomal system. Malfunction of human TPC1 or TPC2 (hTPC) results in severe organellar storage diseases and membrane trafficking defects. Here, we compared the lipid-binding characteristics of hTPC2 and of the PI(3,5)P2-insensitive TPC1 from the model plant Arabidopsis thaliana. Combination of simulations with functional analysis of channel mutants revealed the presence of an hTPC2-specific lipid-binding pocket mutually formed by two channel regions exposed to the cytosolic side of the membrane. We showed that PI(3,5)P2 is simultaneously stabilized by positively charged amino acids (K203, K204, and K207) in the linker between transmembrane helices S4 and S5 and by S322 in the cytosolic extension of S6. We suggest that PI(3,5)P2 cross links two parts of the channel, enabling their coordinated movement during channel gating.

AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling.

Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner. This depolarization is strongly impaired in aux1 mutants, indicating that AUX1 is the major transporter for auxin uptake in root hairs. Local intracellular auxin application triggers Ca2+ signals that propagate as long-distance waves between root cells and modulate their auxin responses. AUX1-mediated IAA transport, as well as IAA- triggered calcium signals, are blocked by treatment with the SCFTIR1/AFB - inhibitor auxinole. Further, they are strongly reduced in the tir1afb2afb3 and the cngc14 mutant. Our study reveals that the AUX1 transporter, the SCFTIR1/AFB receptor and the CNGC14 Ca2+ channel, mediate fast auxin signaling in roots.

BiFC Assay to Detect Calmodulin Binding to Plant Receptor Kinases.

Plant receptor-like kinases (RLKs) are regulated at various levels including posttranscriptional modification and interaction with regulatory proteins. Calmodulin (CaM) is a calcium-sensing protein that was shown to bind to some RLKs such as the PHYTOSULFOKINE RECEPTOR1 (PSKR1). The CaM-binding site is embedded in subdomain VIa of the kinase domain. It is possible that many more of RLKs interact with CaM than previously described. To unequivocally confirm CaM binding, several methods exist. Bimolecular fluorescence complementation (BiFC) and pull-down assays have been successfully used to study CaM binding to PSKR1 and are described in this chapter (BiFC) and in Chapter 15 (pull down). The two methods are complementary. BiFC is useful to show localization and interaction of soluble as well as of membrane-bound proteins in planta.

The phosphoinositide PI(3,5)P2 mediates activation of mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast and plant cells.

Two-pore channel proteins (TPC) encode intracellular ion channels in both animals and plants. In mammalian cells, the two isoforms (TPC1 and TPC2) localize to the endo-lysosomal compartment, whereas the plant TPC1 protein is targeted to the membrane surrounding the large lytic vacuole. Although it is well established that plant TPC1 channels activate in a voltage- and calcium-dependent manner in vitro, there is still debate on their activation under physiological conditions. Likewise, the mode of animal TPC activation is heavily disputed between two camps favoring as activator either nicotinic acid adenine dinucleotide phosphate (NAADP) or the phosphoinositide PI(3,5)P2. Here, we investigated TPC current responses to either of these second messengers by whole-vacuole patch-clamp experiments on isolated vacuoles of Arabidopsis thaliana. After expression in mesophyll protoplasts from Arabidopsis tpc1 knock-out plants, we detected the Arabidopsis TPC1-EGFP and human TPC2-EGFP fusion proteins at the membrane of the large central vacuole. Bath (cytosolic) application of either NAADP or PI(3,5)P2 did not affect the voltage- and calcium-dependent characteristics of AtTPC1-EGFP. By contrast, PI(3,5)P2 elicited large sodium currents in hTPC2-EGFP-containing vacuoles, while NAADP had no such effect. Analogous results were obtained when PI(3,5)P2 was applied to hTPC2 expressed in baker's yeast giant vacuoles. Our results underscore the fundamental differences in the mode of current activation and ion selectivity between animal and plant TPC proteins and corroborate the PI(3,5)P2-mediated activation and Na(+) selectivity of mammalian TPC2.

Novel PSI domains in plant and animal H+-inositol symporters.

Inositols are indispensable components of cellular signaling molecules, and impaired cytoplasmic inositol concentrations affect cellular development. Although most cells can synthesize inositol de novo, plasma membrane-localized inositol uptake systems are indispensable for normal development. Here, we present in-depth functional analyses of plasma membrane-localized H(+)-inositol symporters from human and from the higher plant Arabidopsis thaliana. Sequence comparisons, structural and phylogenetic analyses revealed that these transporters possess conserved extracellular loop domains that represent homologs of plexins/semaphorin/integrin (PSI) domains from animal type I receptors. In these receptors, PSI domains modulate intracellular signaling via extracellular protein-protein interactions. Comparisons of H(+)-inositol symporters with wild type, mutated and truncated PSI domains in different expression systems showed that removal of the entire loop domain increased the V(max) of inositol uptake. Finally, we show that the PSI domains are targets for Ni(++) ions that cause a complete loss of transport activity. A possible role of Ni(++)-binding to PSI domains in Ni(++)-induced carcinogenicity is discussed.

Salt-dependent regulation of a CNG channel subfamily in Arabidopsis.

BACKGROUND: In Arabidopsis thaliana, the family of cyclic nucleotide-gated channels (CNGCs) is composed of 20 members. Previous studies indicate that plant CNGCs are involved in the control of growth processes and responses to abiotic and biotic stresses. According to their proposed function as cation entry pathways these channels contribute to cellular cation homeostasis, including calcium and sodium, as well as to stress-related signal transduction. Here, we studied the expression patterns and regulation of CNGC19 and CNGC20, which constitute one of the five CNGC subfamilies. RESULTS: GUS, GFP and luciferase reporter assays were used to study the expression of CNGC19 and CNGC20 genes from Arabidopsis thaliana in response to developmental cues and salt stress. CNGC19 and CNGC20 were differentially expressed in roots and shoots. The CNGC19 gene was predominantly active in roots already at early growth stages. Major expression was observed in the phloem. CNGC20 showed highest promoter activity in mesophyll cells surrounding the veins. Its expression increased during development and was maximal in mature and senescent leaves. Both genes were upregulated in the shoot in response to elevated NaCl but not mannitol concentrations. While in the root, CNGC19 did not respond to changes in the salt concentration, in the shoot it was strongly upregulated in the observed time frame (6-72 hours). Salt-induction of CNGC20 was also observed in the shoot, starting already one hour after stress treatment. It occurred with similar kinetics, irrespective of whether NaCl was applied to roots of intact plants or to the petiole of detached leaves. No differences in K and Na contents of the shoots were measured in homozygous T-DNA insertion lines for CNGC19 and CNGC20, respectively, which developed a growth phenotype in the presence of up to 75 mM NaCl similar to that of the wild type. CONCLUSION: Together, the results strongly suggest that both channels are involved in the salinity response of different cell types in the shoot. Upon salinity both genes are upregulated within hours. CNGC19 and CNGC20 could assist the plant to cope with toxic effects caused by salt stress, probably by contributing to a re-allocation of sodium within the plant.

Arabidopsis INOSITOL TRANSPORTER2 mediates H+ symport of different inositol epimers and derivatives across the plasma membrane.

Of the four genes of the Arabidopsis (Arabidopsis thaliana) INOSITOL TRANSPORTER family (AtINT family) so far only AtINT4 has been described. Here we present the characterization of AtINT2 and AtINT3. cDNA sequencing revealed that the AtINT3 gene is incorrectly spliced and encodes a truncated protein of only 182 amino acids with four transmembrane helices. In contrast, AtINT2 codes for a functional transporter. AtINT2 localization in the plasma membrane was demonstrated by transient expression of an AtINT2-GREEN FLUORESCENT PROTEIN fusion in Arabidopsis and tobacco (Nicotiana tabacum) epidermis cells and in Arabidopsis protoplasts. Its functional and kinetic properties were determined by expression in yeast (Saccharomyces cerevisiae) cells and Xenopus laevis oocytes. Expression of AtINT2 in a Deltaitr1 (inositol uptake)/Deltaino1 (inositol biosynthesis) double mutant of bakers' yeast complemented the deficiency of this mutant to grow on low concentrations of myoinositol. In oocytes, AtINT2 mediated the symport of H(+) and several inositol epimers, such as myoinositol, scylloinositol, d-chiroinositol, and mucoinositol. The preference for individual epimers differed from that found for AtINT4. Moreover, AtINT2 has a lower affinity for myoinositol (K(m) = 0.7-1.0 mm) than AtINT4 (K(m) = 0.24 mm), and the K(m) is slightly voltage dependent, which was not observed for AtINT4. Organ and tissue specificity of AtINT2 expression was analyzed in AtINT2 promoter/reporter gene plants and showed weak expression in the anther tapetum, the vasculature, and the leaf mesophyll. A T-DNA insertion line (Atint2.1) and an Atint2.1/Atint4.2 double mutant were analyzed under different growth conditions. The physiological roles of AtINT2 are discussed.

Ca2+-dependent and -independent abscisic acid activation of plasma membrane anion channels in guard cells of Nicotiana tabacum.

Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca(2+) concentration. In Vicia faba, however, guard cell anion channels activate in a Ca(2+)-independent manner. Because of potential differences between species, Nicotiana tabacum guard cells were studied in intact plants, with simultaneous recordings of the plasma membrane conductance and the cytoplasmic free Ca(2+) concentration. ABA triggered transient rises in cytoplasmic Ca(2+) in the majority of the guard cells (14 out of 19). In seven out of 14 guard cells, the change in cytoplasmic free Ca(2+) closely matched the activation of anion channels, while the Ca(2+) rise was delayed in seven other cells. In the remaining five cells, ABA stimulated anion channels without a change in the cytoplasmic Ca(2+) level. Even though ABA could activate anion channels in N. tabacum guard cells independent of a rise in the cytoplasmic Ca(2+) concentration, patch clamp experiments showed that anion channels in these cells are stimulated by elevated Ca(2+) in an ATP-dependent manner. Guard cells thus seem to have evolved both Ca(2+)-independent and -dependent ABA signaling pathways. Guard cells of N. tabacum apparently utilize both pathways, while ABA signaling in V. faba seems to be restricted to the Ca(2+)-independent pathway.

Parallel recordings of photosynthetic electron transport and K+-channel activity in single guard cells.

Stomata open in response to red and blue light. Red light-induced stomatal movement is mediated by guard cell chloroplasts and related to K+-uptake into these motor cells. We have combined a new type of microchlorophyll fluorometer with the patch-clamp technique for parallel studies of the photosynthetic electron transport and activity of plasma membrane K+ channels in single guard cell protoplast. In the whole-cell configuration and presence of ATP in the patch-pipette, the activity of the K+-uptake channels remained constant throughout the course of an experiment (up to 30 min) while photosynthetic activity declined to about 50%. In the absence of ATP inward K+ currents declined in a time-dependent manner. Under these ATP-free conditions, photosynthetic electron transport was completely blocked within 8 min. ADP together with orthophosphate was able to prevent inhibition of photosynthetic electron transport and run-down of K+-channel activity. The results demonstrate that the combination of these two techniques is suited to directly study cytosolic factors as common regulators of photosynthesis and plasma membrane transport within a single-cell.