It is generally accepted that K ؉ uptake into guard cells via inwardrectifying K ؉ channels is required for stomatal opening. To test whether the guard cell K ؉ channel KAT1 is essential for stomatal opening, a knockout mutant, KAT1::En-1, was isolated from an En-1 mutagenized Arabidopsis thaliana population. Stomatal action and K ؉ uptake, however, were not impaired in KAT1-deficient plants. Reverse transcription-PCR experiments with isolated guard cell protoplasts showed that in addition to KAT1, the K ؉ channels AKT1, AKT2͞3, AtKC1, and KAT2 were expressed in this cell type. In impalement measurements, intact guard cells exhibited inwardrectifying K ؉ currents across the plasma membrane of both wildtype and KAT1::En-1 plants. This study demonstrates that multiple K ؉ channel transcripts exist in guard cells and that KAT1 is not essential for stomatal action.

Ion channels in roots allow the plant to gain access to nutrients. The composition of the individual ion channels and the functional contribution of different ␣-subunits is largely unknown. Focusing on K ؉ -selective ion channels, we have characterized AtKC1, a new ␣-subunit from the Arabidopsis shaker-like ion channel family. Promoter-␤-glucuronidase (GUS) studies identified AtKC1 expression predominantly in root hairs and root endodermis. Specific antibodies recognized AtKC1 at the plasma membrane. To analyze further the abundance and the functional contribution of the different K ؉ channels ␣-subunits in root cells, we performed real-time reverse transcription-PCR and patch-clamp experiments on isolated root hair protoplasts. Studying all shaker-like ion channel ␣-subunits, we only found the K ؉ inward rectifier AtKC1 and AKT1 and the K ؉ outward rectifier GORK to be expressed in this cell type. Akt1 knockout plants essentially lacked inward rectifying K ؉ currents. In contrast, inward rectifying K ؉ currents were present in AtKC1 knockout plants, but fundamentally altered with respect to gating and cation sensitivity. This indicates that the AtKC1 ␣-subunit represents an integral component of functional root hair K ؉ uptake channels.

Bactericidal/permeability-increasing protein [BPI] is a cationic antimicrobial protein from neutrophils that specifically binds to the surfaces of Gram-negative bacteria via the lipid A component of lipopolysaccharide. To obtain information about the responses of Salmonella typhimurium to cell-surface damage by BPI, two-dimensional gel electrophoresis and N-terminal microsequencing were used to identify proteins that were induced or repressed following BPI treatment. The majority of the affected proteins are involved in central metabolic processes. Upon addition of BPI, the beta-subunit of the F1 portion of Escherichia coli ATP synthase was repressed threefold whereas six proteins were induced up to 11-fold. Three of the latter were identified as lipoamide dehydrogenase, enoyl-acyl carrier protein reductase, and the heat-shock protein HtpG. Additionally, a novel protein, BipA, was identified that is induced over sevenfold by BPI; sequence analysis suggests that it belongs to the GTPase superfamily and interacts with ribosomes. A conserved direct-repeat motif is present in the regulatory regions of several BPI-inducible genes, including the bipA gene. Only one of the BPI-responsive proteins was induced when cells were treated with polymyxin B, which also binds to lipid A. We therefore conclude that BPI and polymyxin B affect different global regulatory networks in S. typhimurium even though they bind with high affinity to the same cell-surface component.

FcRn is a key player in several immunological and non-immunological processes, as it mediates maternal-fetal transfer of IgG, regulates the serum persistence of IgG and albumin, and transports both ligands between different cellular compartments. In addition, FcRn enhances antigen presentation. Thus, there is an intense interest in studies of how FcRn binds and transports its cargo within and across several types of cells, and FcRn detection reagents are in high demand. Here we report on phage display-selected Nanobodies that target human FcRn. The Nanobodies were obtained from a variable-domain repertoire library isolated from a llama immunized with recombinant human FcRn. One candidate, Nb218-H4, was shown to bind FcRn with high affinity at both acidic and neutral pH, without competing ligand binding and interfering with FcRn functions, such as transcytosis of IgG. Thus, Nb218-H4 can be used as a detection probe and as a tracker for visualization of FcRn-mediated cellular transport.