Serine 937 phosphorylation enhances KCC2 activity and strengthens synaptic inhibition.

The potassium chloride cotransporter KCC2 is crucial for Cl- extrusion from mature neurons and thus key to hyperpolarizing inhibition. Auditory brainstem circuits contain well-understood inhibitory projections and provide a potent model to study the regulation of synaptic inhibition. Two peculiarities of the auditory brainstem are (i) posttranslational activation of KCC2 during development and (ii) extremely negative reversal potentials in specific circuits. To investigate the role of the potent phospho-site serine 937 therein, we generated a KCC2 Thr934Ala/Ser937Asp double mutation, in which Ser937 is replaced by aspartate mimicking the phosphorylated state, and the neighbouring Thr934 arrested in the dephosphorylated state. This double mutant showed a twofold increased transport activity in HEK293 cells, raising the hypothesis that auditory brainstem neurons show lower [Cl-]i. and increased glycinergic inhibition. This was tested in a mouse model carrying the same KCC2 Thr934Ala/Ser937Asp mutation by the use of the CRISPR/Cas9 technology. Homozygous KCC2 Thr934Ala/Ser937Asp mice showed an earlier developmental onset of hyperpolarisation in the auditory brainstem. Mature neurons displayed stronger glycinergic inhibition due to hyperpolarized ECl-. These data demonstrate that phospho-regulation of KCC2 Ser937 is a potent way to interfere with the excitation-inhibition balance in neural circuits.

K+/Cl- cotransporter 2 (KCC2) and Na+/HCO3- cotransporter 1 (NBCe1) interaction modulates profile of KCC2 phosphorylation.

K+/Cl- cotransporter 2 (KCC2) is a major Cl- extruder in mature neurons and is responsible for the establishment of low intracellular [Cl-], necessary for fast hyperpolarizing GABAA-receptor mediated synaptic inhibition. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a pH regulatory protein expressed in neurons and glial cells. An interactome study identified NBCe1 as a possible interaction partner of KCC2. In this study, we investigated the putative effect of KCC2/NBCe1 interaction in baseline and the stimulus-induced phosphorylation pattern and function of KCC2. Primary mouse hippocampal neuronal cultures from wildtype (WT) and Nbce1-deficient mice, as well as HEK-293 cells stably transfected with KCC2WT, were used. The results show that KCC2 and NBCe1 are interaction partners in the mouse brain. In HEKKCC2 cells, pharmacological inhibition of NBCs with S0859 prevented staurosporine- and 4-aminopyridine (4AP)-induced KCC2 activation. In mature cultures of hippocampal neurons, however, S0859 completely inhibited postsynaptic GABAAR and, thus, could not be used as a tool to investigate the role of NBCs in GABA-dependent neuronal networks. In Nbce1-deficient immature hippocampal neurons, baseline phosphorylation of KCC2 at S940 was downregulated, compared to WT, and exposure to staurosporine failed to reduce pKCC2 S940 and T1007. In Nbce1-deficient mature neurons, baseline levels of pKCC2 S940 and T1007 were upregulated compared to WT, whereas after 4AP treatment, pKCC2 S940 was downregulated, and pKCC2 T1007 was further upregulated. Functional experiments showed that the levels of GABAAR reversal potential, baseline intracellular [Cl-], Cl- extrusion, and baseline intracellular pH were similar between WT and Nbce1-deficient neurons. Altogether, our data provide a primary description of the properties of KCC2/NBCe1 protein-protein interaction and implicate modulation of stimulus-mediated phosphorylation of KCC2 by NBCe1/KCC2 interaction-a mechanism with putative pathophysiological relevance.

Both chloride-binding sites are required for KCC2-mediated transport.

The K+-Cl- cotransporter 2 (KCC2) plays an important role in inhibitory neurotransmission, and its impairment is associated with neurological and psychiatric disorders, including epilepsy, schizophrenia, and autism. Although KCCs transport K+ and Cl- in a 1:1 stoichiometry, two Cl- coordination sites were indicated via cryo-EM. In a comprehensive analysis, we analyzed the consequences of point mutations of residues coordinating Cl- in Cl1 and Cl2. Individual mutations of residues in Cl1 and Cl2 reduce or abolish KCC2WT function, indicating a crucial role of both Cl- coordination sites for KCC2 function. Structural changes in the extracellular loop 2 by inserting a 3xHA tag switches the K+ coordination site to another position. To investigate, whether the extension of the extracellular loop 2 with the 3xHA tag also affects the coordination of the two Cl- coordination sites, we carried out the analogous experiments for both Cl- coordinating sites in the KCC2HA construct. These analyses showed that most of the individual mutation of residues in Cl1 and Cl2 in the KCC2HA construct reduces or abolishes KCC2 function, indicating that the coordination of Cl- remains at the same position. However, the coupling of K+ and Cl- in Cl1 is still apparent in the KCC2HA construct, indicating a mutual dependence of both ions. In addition, the coordination residue Tyr569 in Cl2 shifted in KCC2HA. Thus, conformational changes in the extracellular domain affect K+ and Cl--binding sites. However, the effect on the Cl--binding sites is subtler.

NKCC1 and KCC2: Structural insights into phospho-regulation.

Inhibitory neurotransmission plays a fundamental role in the central nervous system, with about 30-50% of synaptic connections being inhibitory. The action of both inhibitory neurotransmitter, gamma-aminobutyric-acid (GABA) and glycine, mainly relies on the intracellular Cl- concentration in neurons. This is set by the interplay of the cation chloride cotransporters NKCC1 (Na+, K+, Cl- cotransporter), a main Cl- uptake transporter, and KCC2 (K+, Cl- cotransporter), the principle Cl- extruder in neurons. Accordingly, their dysfunction is associated with severe neurological, psychiatric, and neurodegenerative disorders. This has triggered great interest in understanding their regulation, with a strong focus on phosphorylation. Recent structural data by cryogenic electron microscopy provide the unique possibility to gain insight into the action of these phosphorylations. Interestingly, in KCC2, six out of ten (60%) known regulatory phospho-sites reside within a region of 134 amino acid residues (12% of the total residues) between helices α8 and α9 that lacks fixed or ordered three-dimensional structures. It thus represents a so-called intrinsically disordered region. Two further phospho-sites, Tyr903 and Thr906, are also located in a disordered region between the ß8 strand and the α8 helix. We make the case that especially the disordered region between helices α8 and α9 acts as a platform to integrate different signaling pathways and simultaneously constitute a flexible, highly dynamic linker that can survey a wide variety of distinct conformations. As each conformation can have distinct binding affinities and specificity properties, this enables regulation of [Cl-] i and thus the ionic driving force in a history-dependent way. This region might thus act as a molecular processor underlying the well described phenomenon of ionic plasticity that has been ascribed to inhibitory neurotransmission. Finally, it might explain the stunning long-range effects of mutations on phospho-sites in KCC2.

Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion transport.

K+-Cl- cotransporters (KCCs) play important roles in physiological processes such as inhibitory neurotransmission and cell-volume regulation. KCCs exhibit significant variations in K+ affinities, yet recent atomic structures demonstrated that K+- and Cl--binding sites are highly conserved, raising the question of whether additional structural elements may contribute to ion coordination. The termini and the large extracellular domain (ECD) of KCCs exhibit only low sequence identity and were already discussed as modulators of transport activity. Here, we used the extracellular loop 2 (EL2) that links transmembrane helices (TMs) 3 and 4, as a mechanism to modulate ECD folding. We compared consequences of point mutations in the K+-binding site on the function of WT KCC2 and in a KCC2 variant, in which EL2 was structurally altered by insertion of a IFYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHAAA (3xHA) tag (36 amino acids). In WT KCC2, individual mutations of five residues in the K+-binding site resulted in a 2- to 3-fold decreased transport rate. However, the same mutations in the KCC2 variant with EL2 structurally altered by insertion of a 3xHA tag had no effect on transport activity. Homology models of mouse KCC2 with the 3xHA tag inserted into EL2 using ab initio prediction were generated. The models suggest subtle conformational changes occur in the ECD upon EL2 modification. These data suggest that a conformational change in the ECD, for example, by interaction with EL2, might be an elegant way to modulate the K+ affinity of the different isoforms in the KCC subfamily.

Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1.

The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies. Our analyses revealed that application of staurosporine or NEM dephosphorylates Thr1007 of KCC2, and Thr203, Thr207 and Thr212 of NKCC1. Dephosphorylation of Thr1007 of KCC2, and Thr207 and Thr212 of NKCC1 were previously demonstrated to activate KCC2 and to inactivate NKCC1. In addition, application of the two agents resulted in dephosphorylation of the T-loop and S-loop phosphorylation sites Thr233 and Ser373 of SPAK, a critical kinase in the WNK-SPAK/OSR1 signaling module mediating phosphorylation of KCC2 and NKCC1. Taken together, these results suggest that reciprocal regulation of KCC2 and NKCC1 via staurosporine and NEM is based on WNK-SPAK/OSR1 signaling. The key regulatory phospho-site Ser940 of KCC2 is not critically involved in the enhanced activation of KCC2 upon staurosporine and NEM treatment, as both agents have opposite effects on its phosphorylation status. Finally, NEM acts in a tissue-specific manner on Ser940, as shown by comparative analysis in HEK293 cells and immature cultured hippocampal neurons. In summary, our analyses identified phospho-sites that are responsive to staurosporine or NEM application. This provides important information towards a better understanding of the cooperative interactions of different phospho-sites.

Phosphoregulation of the intracellular termini of K+-Cl- cotransporter 2 (KCC2) enables flexible control of its activity.

The pivotal role of K+-Cl- cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five bona fide phosphosites Ser31, Thr34, Ser932, Thr999, and Thr1008 was investigated by the use of alanine and aspartate mutants. Tl+-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent. Increased activity was due to changes in intrinsic activity, as it was not caused by increased cell-surface abundance. Substitutions of Ser31, Thr34, or Thr999 had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and N-ethylmaleimide (NEM) involved multiple phosphosites. S31D, T34A, S932A/D, T999A, or T1008A/D abrogated staurosporine mediated stimulation, and S31A, T34D, or S932D abolished NEM-mediated stimulation. This demonstrates for the first time differential effects of staurosporine and NEM on KCC2. In addition, the staurosporine-mediated effects involved both KCC2 phosphorylation and dephosphorylation with Ser932 and Thr1008 being bona fide target sites. In summary, our data reveal a complex phosphoregulation of KCC2 that provides the transporter with a toolbox for graded activity and integration of different signaling pathways.

Molecular cloning and biochemical characterization of two cation chloride cotransporter subfamily members of Hydra vulgaris.

Cation Chloride Cotransporters (CCCs) comprise secondary active membrane proteins mainly mediating the symport of cations (Na+, K+) coupled with chloride (Cl-). They are divided into K+-Cl- outward transporters (KCCs), the Na+-K+-Cl- (NKCCs) and Na+-Cl- (NCCs) inward transporters, the cation chloride cotransporter interacting protein CIP1, and the polyamine transporter CCC9. KCCs and N(K)CCs are established in the genome since eukaryotes and metazoans, respectively. Most of the physiological and functional data were obtained from vertebrate species. To get insights into the basal functional properties of KCCs and N(K)CCs in the metazoan lineage, we cloned and characterized KCC and N(K)CC from the cnidarian Hydra vulgaris. HvKCC is composed of 1,032 amino-acid residues. Functional analyses revealed that hvKCC mediates a Na+-independent, Cl- and K+ (Tl+)-dependent cotransport. The classification of hvKCC as a functional K-Cl cotransporter is furthermore supported by phylogenetic analyses and a similar structural organization. Interestingly, recently obtained physiological analyses indicate a role of cnidarian KCCs in hyposmotic volume regulation of nematocytes. HvN(K)CC is composed of 965 amino-acid residues. Phylogenetic analyses and structural organization suggest that hvN(K)CC is a member of the N(K)CC subfamily. However, no inorganic ion cotransport function could be detected using different buffer conditions. Thus, hvN(K)CC is a N(K)CC subfamily member without a detectable inorganic ion cotransporter function. Taken together, the data identify two non-bilaterian solute carrier 12 (SLC12) gene family members, thereby paving the way for a better understanding of the evolutionary paths of this important cotransporter family.

Molecular bases of K+ secretory cells in the inner ear: shared and distinct features between birds and mammals.

In the cochlea, mammals maintain a uniquely high endolymphatic potential (EP), which is not observed in other vertebrate groups. However, a high [K+] is always present in the inner ear endolymph. Here, we show that Kir4.1, which is required in the mammalian stria vascularis to generate the highly positive EP, is absent in the functionally equivalent avian tegmentum vasculosum. In contrast, the molecular repertoire required for K+ secretion, specifically NKCC1, KCNQ1, KCNE1, BSND and CLC-K, is shared between the tegmentum vasculosum, the vestibular dark cells and the marginal cells of the stria vascularis. We further show that in barn owls, the tegmentum vasculosum is enlarged and a higher EP (~+34 mV) maintained, compared to other birds. Our data suggest that both the tegmentum vasculosum and the stratified stria vascularis evolved from an ancestral vestibular epithelium that already featured the major cell types of the auditory epithelia. Genetic recruitment of Kir4.1 specifically to strial melanocytes was then a crucial step in mammalian evolution enabling an increase in the cochlear EP. An increased EP may be related to high-frequency hearing, as this is a hallmark of barn owls among birds and mammals among amniotes.

Neto2-null mice have impaired GABAergic inhibition and are susceptible to seizures.

Neto2 is a transmembrane protein that interacts with the neuron-specific K(+)-Cl(-) cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl(-) gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter. In the present study, we characterized GABAergic inhibition and KCC2-mediated neuronal chloride homeostasis in pyramidal neurons from adult hippocampal slices. Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized. We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2(-/-) neurons compared to wild-type controls. To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2(-/-) neurons had significant reductions in both their amplitude and frequency. Based on the critical role of Neto2 in regulating GABAergic inhibition we rationalized that Neto2-null mice would be prone to seizure activity. We found that Neto2-null mice demonstrated a decrease in the latency to pentylenetetrazole (PTZ)-induced seizures and an increase in seizure severity.

A novel regulatory locus of phosphorylation in the C terminus of the potassium chloride cotransporter KCC2 that interferes with N-ethylmaleimide or staurosporine-mediated activation.

The neuron-specific cation chloride cotransporter KCC2 plays a crucial role in hyperpolarizing synaptic inhibition. Transporter dysfunction is associated with various neurological disorders, raising interest in regulatory mechanisms. Phosphorylation has been identified as a key regulatory process. Here, we retrieved experimentally observed phosphorylation sites of KCC2 from public databases and report on the systematic analysis of six phosphorylated serines, Ser(25), Ser(26), Ser(937), Ser(1022), Ser(1025), and Ser(1026). Alanine or aspartate substitutions of these residues were analyzed in HEK-293 cells. All mutants were expressed in a pattern similar to wild-type KCC2 (KCC2(WT)). Tl(+) flux measurements demonstrated unchanged transport activity for Ser(25), Ser(26), Ser(1022), Ser(1025), and Ser(1026) mutants. In contrast, KCC2(S937D), mimicking phosphorylation, resulted in a significant up-regulation of transport activity. Aspartate substitution of Thr(934), a neighboring putative phosphorylation site, resulted in a comparable increase in KCC2 transport activity. Both KCC2(T934D) and KCC2(S937D) mutants were inhibited by the kinase inhibitor staurosporine and by N-ethylmaleimide, whereas KCC2(WT), KCC2(T934A), and KCC2(S937A) were activated. The inverse staurosporine effect on aspartate versus alanine substitutions reveals a cross-talk between different phosphorylation sites of KCC2. Immunoblot and cell surface labeling experiments detected no alterations in total abundance or surface expression of KCC2(T934D) and KCC2(S937D) compared with KCC2(WT). These data reveal kinetic regulation of transport activity by these residues. In summary, our data identify a novel key regulatory phosphorylation site of KCC2 and a functional interaction between different conformation-changing post-translational modifications. The action of pharmacological agents aimed to modulate KCC2 activity for therapeutic benefit might therefore be highly context-specific.

Evolution of the cation chloride cotransporter family: ancient origins, gene losses, and subfunctionalization through duplication.

The cation chloride cotransporter (CCCs) family comprises of four subfamilies-K(+)-Cl(-) cotransporters (KCCs), Na(+)-K(+)-2Cl(-) cotransporters (NKCCs), and Na(+)-Cl(-) cotransporters (NCCs)-and possibly two additional members-CCC interacting protein (CIP1) and polyamine transporters (CCC9)-as well. Altogether, CCCs can play essential physiological roles in transepithelial ion reabsorption and secretion, cell volume regulation, and inhibitory neurotransmission and so are present across all domains of life. To gain insight into the evolution of this family, we performed a comprehensive phylogenetic analysis using publically available genomic information. Our results clearly support CIP1 as being a true CCC based on shared evolutionary history. By contrast, the status of CCC9 in this regard remains equivocal. We also reveal the existence of a single ancestral CCC gene present in Archaea, from which numerous duplication events at the base of archaeans and eukaryotes lead to the divergence and subsequent neofunctionalization of the paralogous CCC subfamilies. A diversity of ensuing gene-loss events resulted in the complex distribution of CCCs present across the different taxa. Importantly, the occurrence of KCCs in "basal" metazoan taxa like sponges would allow an early formation of fast hyperpolarizing neurotransmission in metazoans. Gene duplications within the CCC subfamilies in vertebrates (in particular, KCCs, NKCCs, and NCCs) lend further evidence to the 2R hypothesis of two rounds of genome duplication at the base of the vertebrate lineage, especially in concert with our syntenic cluster analyses. This increased number of KCCs, NKCCs, and NCCs isoforms facilitates their further, important subfunctionalization in the vertebrate lineage.

Molecular and evolutionary insights into the structural organization of cation chloride cotransporters.

Cation chloride cotransporters (CCC) play an essential role for neuronal chloride homeostasis. K(+)-Cl(-) cotransporter (KCC2), is the principal Cl(-)-extruder, whereas Na(+)-K(+)-Cl(-) cotransporter (NKCC1), is the major Cl(-)-uptake mechanism in many neurons. As a consequence, the action of the inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine strongly depend on the activity of these two transporters. Knowledge of the mechanisms involved in ion transport and regulation is thus of great importance to better understand normal and disturbed brain function. Although no overall 3-dimensional crystal structures are yet available, recent molecular and phylogenetic studies and modeling have provided new and exciting insights into structure-function relationships of CCC. Here, we will summarize our current knowledge of the gross structural organization of the proteins, their functional domains, ion binding and translocation sites, and the established role of individual amino acids (aa). A major focus will be laid on the delineation of shared and distinct organizational principles between KCC2 and NKCC1. Exploiting the richness of recently generated genome data across the tree of life, we will also explore the molecular evolution of these features.

Egr2::cre mediated conditional ablation of dicer disrupts histogenesis of mammalian central auditory nuclei.

Histogenesis of the auditory system requires extensive molecular orchestration. Recently, Dicer1, an essential gene for generation of microRNAs, and miR-96 were shown to be important for development of the peripheral auditory system. Here, we investigated their role for the formation of the auditory brainstem. Egr2::Cre-mediated early embryonic ablation of Dicer1 caused severe disruption of auditory brainstem structures. In adult animals, the volume of the cochlear nucleus complex (CNC) was reduced by 73.5%. This decrease is in part attributed to the lack of the microneuronal shell. In contrast, fusiform cells, which similar to the granular cells of the microneural shell are derived from Egr2 positive cells, were still present. The volume reduction of the CNC was already present at birth (67.2% decrease). The superior olivary complex was also drastically affected in these mice. Nissl staining as well as Vglut1 and Calbindin 1 immunolabeling revealed that principal SOC nuclei such as the medial nucleus of the trapezoid body and the lateral superior olive were absent. Only choline acetyltransferase positive neurons of the olivocochlear bundle were observed as a densely packed cell group in the ventrolateral area of the SOC. Mid-embryonic ablation of Dicer1 in the ventral cochlear nucleus by Atoh7::Cre-mediated recombination resulted in normal formation of the cochlear nucleus complex, indicating an early embryonic requirement of Dicer1. Quantitative RT-PCR analysis of miR-96 demonstrated low expression in the embryonic brainstem and up-regulation thereafter, suggesting that other microRNAs are required for proper histogenesis of the auditory brainstem. Together our data identify a critical role of Dicer activity during embryonic development of the auditory brainstem.

KCC2 transport activity requires the highly conserved L675 in the C-terminal ß1 strand.

The activity of the neuron-specific K(+), Cl(-) co-transporter 2 (KCC2) is required for hyperpolarizing action of GABA and glycine. KCC2-mediated transport therefore plays a pivotal role in neuronal inhibition. Few analyses have addressed the amino acid requirements for transport-competent conformation. KCC2 consists of 12 transmembrane domains flanked by two intracellular termini. Structural analyses of a related archaeal protein have identified an evolutionary extremely conserved ß1 strand, which links the transmembrane domain to a C-terminal dimerization interface. Here, we focused on the sequence requirement of this linker. We mutated four highly conserved amino acids of the ß1 strand ((673)QLLV(676)) to alanine and analyzed the functional consequences in mammalian cells. Flux measurements demonstrated that L(675A) significantly reduced KCC2 transport activity by 41%, whereas the other three mutants displayed normal activity. Immunocytochemistry and cell surface labeling revealed normal trafficking of all four mutants. Altogether, our results identify L(675) as a critical residue for KCC2 transport activity. Furthermore, in view of its evolutionary conservation, the data suggest a remarkable tolerance of the KCC2 transport activity to amino acid substitutions in the ß1 strand.

Opposite temperature effect on transport activity of KCC2/KCC4 and N(K)CCs in HEK-293 cells.

BACKGROUND: Cation chloride cotransporters play essential roles in many physiological processes such as volume regulation, transepithelial salt transport and setting the intracellular chloride concentration in neurons. They consist mainly of the inward transporters NCC, NKCC1, and NKCC2, and the outward transporters KCC1 to KCC4. To gain insight into regulatory and structure-function relationships, precise determination of their activity is required. Frequently, these analyses are performed in HEK-293 cells. Recently the activity of the inward transporters NKCC1 and NCC was shown to increase with temperature in these cells. However, the temperature effect on KCCs remains largely unknown. FINDINGS: Here, we determined the temperature effect on KCC2 and KCC4 transport activity in HEK-293 cells. Both transporters demonstrated significantly higher transport activity (2.5 fold for KCC2 and 3.3 fold for KCC4) after pre-incubation at room temperature compared to 37°C. CONCLUSIONS: These data identify a reciprocal temperature dependence of cation chloride inward and outward cotransporters in HEK-293 cells. Thus, lower temperature should be used for functional characterization of KCC2 and KCC4 and higher temperatures for N(K)CCs in heterologous mammalian expression systems. Furthermore, if this reciprocal effect also applies to neurons, the action of inhibitory neurotransmitters might be more affected by changes in temperature than previously thought.

Seasonal variation of the effect of extremely diluted agitated gibberellic acid (10e-30) on wheat stalk growth: a multiresearcher study.

The influence of a homeopathic high dilution of gibberellic acid on wheat growth was studied at different seasons of the year. Seedlings were allowed to develop under standardized conditions for 7 days; plants were harvested and stalk lengths were measured. The data obtained confirm previous findings, that ultrahigh diluted potentized gibberellic acid affects stalk growth. Furthermore, the outcome of the study suggests that experiments utilizing the bioassay presented should best be performed in autumn season. In winter and spring, respectively, no reliable effects were found.

Variação sazonal do efeito do ácido giberélico altamente diluído (10-30) sobre o crescimento do trigo - um estudo com vários pesquisadores / Seasonal variation of the effect of extremely diluted agitated gibberellic acid (10-30) on wheat stalk growth - a multi researcher study

Control experiments were performed at different seasons of the year as a follow-up to pilot experiments [1] where a homeopathic high dilution of gibberellic acid had influenced growth in a wheat bio assay (7 days). Grains of winter wheat (Triticum aestivum, Capo variety) were observed under the influence of extremely diluted gibberellic acid (10-30) prepared by stepwise dilution and agitation according to a protocol derived from homeopathy (“G30x”). Analogously prepared water was used for control (“W30x”). Following up on 5 pilot experiments (4 in autumn 2007, 1 in spring 2008), 10 experiments were performed (5 in autumn 2008 or 2009 and 5 in winter 2009 or 2010) with a total of 9 experiments in autumn season (5 researchers, about 9,000 grains), and 6 in winter/spring (4 researchers, about 6,000 grains).(AU)

Experimentos controlados foram realizados em diferentes estações do ano, em continuação a um experimento-piloto [1] onde o ácido giberélico altamente diluído e agitado influenciaram o crescimento de trigo, em um ensaio biológico (7 dias). Grão de trigo de inverno (Triticum aestivum, variedade Capo) foram observados sob a influência do ácido giberélico altamente diluído (10-30) preparado por um processo serial de diluição e agitação, de acordo com um protocolo derivado da homeopatia (“G30x”). Analogamente, o mesmo protocol foi usado para preparar a amostra controle, apenas com água (“W30x”). Em continuidade a realização de 5 experimentos-piloto (4 no outono de 2007, 1 na primavera de 2008), 10 experimentos foram realizados (5 no outono de 2008 ou 2009 e 5 no inverno de 2009 ou 2010) com um total de 9 experimentos na estação do outono (5 pesquisadores, aproximadamente 9.000 grãos) e 6 no inverno/primavera (4 pesquisadores, aproximadamente 6.000 grãos).(AU)