Structure and topography of the synaptic V-ATPase-synaptophysin complex.

Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single-particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the proton gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogues synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin-knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin-knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.

Vph2 is required for protection against a reductive stress in Candida albicans.

Vph2 is a putative V-ATPase assembly factor. Our previous study has characterized its roles in localization of V-ATPase subunit, cell wall composition, hyphal development and virulence. In this study, our results further demonstrated that Vph2 was localized around the nucleus and in patches close to the periphery of the cell, indicating that Vph2 was located to the endoplasmic reticulum (ER), which was consistent with that in Saccharomyces cerevisiae. Disruption of VPH2 led to hypersensitivity to reducing stresses induced by dithiothreitol (DTT) and ß-mercaptoethanol (ß-ME), and displayed increased GSH content and up-regulation of unfolded protein response (UPR)-related genes, such as PRB1 and PMT4. However, the induced UPR and growth defect on ß-ME plates of vph2Δ/Δ mutant could be partly alleviated by the GSH-specific scavenger 1-chloro-2, 4-dinitrobenzene (CDNB). These results indicated that loss of VPH2 led to an increase in GSH levels, which induced the UPR and caused the defective growth on reductive stress induced by ß-ME. In summary, Vph2 is necessary to maintain resistance against reductive stresses.

Increased (pro)renin receptor expression in the subfornical organ of hypertensive humans.

The central nervous system plays an important role in essential hypertension in humans and in animal models of hypertension through modulation of sympathetic activity and Na+ and body fluid homeostasis. Data from animal models of hypertension suggest that the renin-angiotensin system in the subfornical organ (SFO) of the brain is critical for hypertension development. We recently reported that the brain (pro)renin receptor (PRR) is a novel component of the brain renin-angiotensin system and could be a key initiator of the pathogenesis of hypertension. Here, we examined the expression level and cellular distribution of PRR in the SFO of postmortem human brains to assess its association with the pathogenesis of human hypertension. Postmortem SFO tissues were collected from hypertensive and normotensive human subjects. Immunolabeling for the PRR and a retrospective analysis of clinical data were performed. We found that human PRR was prominently expressed in most neurons and microglia, but not in astrocytes, in the SFO. Importantly, PRR levels in the SFO were elevated in hypertensive subjects. Moreover, PRR immunoreactivity was significantly correlated with systolic blood pressure but not body weight, age, or diastolic blood pressure. Interestingly, this correlation was independent of antihypertensive drug therapy. Our data indicate that PRR in the SFO may be a key molecular player in the pathogenesis of human hypertension and, as such, could be an important focus of efforts to understand the neurogenic origin of hypertension. NEW & NOTEWORTHY This study provides evidence that, in the subfornical organ of the human brain, the (pro)renin receptor is expressed in neurons and microglia cells but not in astrocytes. More importantly, (pro)renin receptor immunoreactivity in the subfornical organ is increased in hypertensive humans and is significantly correlated with systolic blood pressure.

The expression patterns of aquaporin 9, vacuolar H+-ATPase, and cytokeratin 5 in the epididymis of the common vampire bat.

Desmodus rotundus is a vampire bat species that inhabits Latin America. Some basic aspects of this species' biology are still unknown, as the histophysiological characteristics of the male reproductive tract. Our study has focused on its epididymis, which is an important organ for performing a variety of functions, especially the sperm maturation and storage. The aim of this study was to identify principal, narrow, clear, and basal cells using cell-specific markers such as aquaporin 9 (AQP9), vacuolar H+-ATPase (V-ATPase), and cytokeratin 5 (KRT5). Principal cells were labeled by AQP9 from initial segment to cauda region in their stereocilia. They were shown with a columnar shape, whereas V-ATPase-rich cells were identified with a goblet-shaped body along the entire epididymis, including the initial segment, which were named as clear cells. Pencil-shaped V-ATPase-rich cells (narrow cells) were not detected in the initial segment of the bat epididymis, unlike in the rodent. Basal cells were labeled by KRT5 and were located at the basal portion of the epithelium forming a dense network. However, no basal cells with a luminal-reaching body extension were observed in the bat epididymis. In summary, epithelial cells were identified by their specific markers in the vampire bat epididymis. Principal and basal cells were labeled by AQP9 and KRT5, respectively. Narrow cells were not observed in the vampire bat epididymis, whereas clear cells were identified by V-ATPase labeling along the entire duct in a goblet-shaped body. In addition, no luminal-reaching basal cells were observed in the vampire bat epididymis.

Aedes aegypti Rhesus glycoproteins contribute to ammonia excretion by larval anal papillae.

In larval Aedes aegypti, transcripts of the Rhesus-like glycoproteins AeRh50-1 and AeRh50-2 have been detected in the anal papillae, sites of ammonia (NH3/NH4+) excretion; however, these putative ammonia transporters have not been previously localized or functionally characterized. In this study, we show that the AeRh50s co-immunolocalize with apical V-type H+-ATPase as well as with basal Na+/K+-ATPase in the epithelium of anal papillae. The double-stranded RNA-mediated knockdown of AeRh50-1 and AeRh50-2 resulted in a significant reduction in AeRh50 protein abundance in the anal papillae, and this was coupled to decreased ammonia excretion. The knockdown of AeRh50-1 resulted in decreased hemolymph [NH4+] and pH whereas knockdown of AeRh50-2 had no effect on these parameters. We conclude that the AeRh50s are important contributors to ammonia excretion at the anal papillae of larval A. aegypti, which may be the basis for their ability to inhabit areas with high ammonia levels.

Ammonia excretion in the marine polychaete Eurythoe complanata (Annelida).

Ammonia is a toxic waste product from protein metabolism and needs to be either converted into less toxic molecules or, in the case of fish and aquatic invertebrates, excreted directly as is. In contrast to fish, very little is known regarding the ammonia excretion mechanism and the participating excretory organs in marine invertebrates. In the current study, ammonia excretion in the marine burrowing polychaete Eurythoe complanata was investigated. As a potential site for excretion, the 100-200 µm long, 30-50 µm wide and up to 25 µm thick dentrically branched, well ventilated and vascularized branchiae (gills) were identified. In comparison to the main body, the branchiae showed considerably higher mRNA expression levels of Na+/K+-ATPase, V-type H+-ATPase, cytoplasmic carbonic anhydrase (CA-2), a Rhesus-like protein, and three different ammonia transporters (AMTs). Experiments on the intact organism revealed that ammonia excretion did not occur via apical ammonia trapping, but was regulated by a basolateral localized V-type H+-ATPase, carbonic anhydrase and intracellular cAMP levels. Interestingly, the V-type H+-ATPase seems to play a role in ammonia retention. A 1 week exposure to 1 mmol l-1 NH4Cl (HEA) did not cause a change in ammonia excretion rates, while the three branchial expressed AMTs showed a tendency to be down-regulated. This indicates a shift of function in the branchial ammonia excretion processes under these conditions.

Alignment of cryo-EM movies of individual particles by optimization of image translations.

Direct detector device (DDD) cameras have revolutionized single particle electron cryomicroscopy (cryo-EM). In addition to an improved camera detective quantum efficiency, acquisition of DDD movies allows for correction of movement of the specimen, due to both instabilities in the microscope specimen stage and electron beam-induced movement. Unlike specimen stage drift, beam-induced movement is not always homogeneous within an image. Local correlation in the trajectories of nearby particles suggests that beam-induced motion is due to deformation of the ice layer. Algorithms have already been described that can correct movement for large regions of frames and for >1 MDa protein particles. Another algorithm allows individual <1 MDa protein particle trajectories to be estimated, but requires rolling averages to be calculated from frames and fits linear trajectories for particles. Here we describe an algorithm that allows for individual <1 MDa particle images to be aligned without frame averaging or linear trajectories. The algorithm maximizes the overall correlation of the shifted frames with the sum of the shifted frames. The optimum in this single objective function is found efficiently by making use of analytically calculated derivatives of the function. To smooth estimates of particle trajectories, rapid changes in particle positions between frames are penalized in the objective function and weighted averaging of nearby trajectories ensures local correlation in trajectories. This individual particle motion correction, in combination with weighting of Fourier components to account for increasing radiation damage in later frames, can be used to improve 3-D maps from single particle cryo-EM.

Participation of the extracellular domain in (pro)renin receptor dimerization.

The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin-angiotensin system. (P)RR is known to form a homodimer, but the region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.

Regulated traffic of anion transporters in mammalian Brunner's glands: a role for water and fluid transport.

The Brunner's glands of the proximal duodenum exert barrier functions through secretion of glycoproteins and antimicrobial peptides. However, ion transporter localization, function, and regulation in the glands are less clear. Mapping the subcellular distribution of transporters is an important step toward elucidating trafficking mechanisms of fluid transport in the gland. The present study examined 1) changes in the distribution of intestinal anion transporters and the aquaporin 5 (AQP5) water channel in rat Brunner's glands following second messenger activation and 2) anion transporter distribution in Brunner's glands from healthy and disease-affected human tissues. Cystic fibrosis transmembrane conductance regulator (CFTR), AQP5, sodium-potassium-coupled chloride cotransporter 1 (NKCC1), sodium-bicarbonate cotransporter (NBCe1), and the proton pump vacuolar ATPase (V-ATPase) were localized to distinct membrane domains and in endosomes at steady state. Carbachol and cAMP redistributed CFTR to the apical membrane. cAMP-dependent recruitment of CFTR to the apical membrane was accompanied by recruitment of AQP5 that was reversed by a PKA inhibitor. cAMP also induced apical trafficking of V-ATPase and redistribution of NKCC1 and NBCe1 to the basolateral membranes. The steady-state distribution of AQP5, CFTR, NBCe1, NKCC1, and V-ATPase in human Brunner's glands from healthy controls, cystic fibrosis, and celiac disease resembled that of rat; however, the distribution profiles were markedly attenuated in the disease-affected duodenum. These data support functional transport of chloride, bicarbonate, water, and protons by second messenger-regulated traffic in mammalian Brunner's glands under physiological and pathophysiological conditions.

Tissue-specific ionomotive enzyme activity and K+ reabsorption reveal the rectum as an important ionoregulatory organ in larval Chironomus riparius exposed to varying salinity.

A role for the rectum in the ionoregulatory homeostasis of larval Chironomus riparius was revealed by rearing animals in different saline environments and examining: (1) the spatial distribution and activity of keystone ionomotive enzymes Na(+)-K(+)-ATPase (NKA) and V-type H(+)-ATPase (VA) in the alimentary canal, and (2) rectal K(+) transport with the scanning ion-selective electrode technique (SIET). NKA and VA activity were measured in four distinct regions of the alimentary canal as follows: the combined foregut and anterior midgut, the posterior midgut, the Malpighian tubules and the hindgut. Both enzymes exhibited 10-20 times greater activity in the hindgut relative to all other areas. When larvae were reared in either ion-poor water (IPW) or freshwater (FW), no significant difference in hindgut enzyme activity was observed. However, in larvae reared in brackish water (BW), NKA and VA activity in the hindgut significantly decreased. Immunolocalization of NKA and VA in the hindgut revealed that the bulk of protein was located in the rectum. Therefore, K(+) transport across the rectum was examined using SIET. Measurement of K(+) flux along the rectum revealed a net K(+) reabsorption that was reduced fourfold in BW-reared larvae versus larvae reared in FW or IPW. Inhibition of NKA with ouabain, VA with bafilomycin and K(+) channels with charybdotoxin diminished rectal K(+) reabsorption in FW- and IPW-reared larvae, but not BW-reared larvae. Data suggest that the rectum of C. riparius plays an important role in allowing these larvae to cope with dilute as well as salinated environmental conditions.

Expression and functional role of vacuolar H(+)-ATPase in human hepatocellular carcinoma.

Tumor cells often exist in a hypoxic microenvironment, which produces acidic metabolites. To survive in this harsh environment, tumor cells must exhibit a dynamic cytosolic pH regulatory system. Vacuolar H(+)-adenosine triphosphatase (V-ATPase) is considered to play an important role in the regulation of the acidic microenvironment of some tumors. In this study, we made an investigation on the expression and functional role of V-ATPase in native human hepatocellular carcinoma (HCC). The results showed that the messenger RNA and protein expression levels of V-ATPase subunit ATP6L in native human HCC tissues were markedly increased, compared with normal liver tissues. Immunohistochemical analysis further confirmed the enhanced expression of V-ATPase ATP6L in human HCC cells and revealed that V-ATPase ATP6L was distributed in the cytoplasm and plasma membrane of HCC cells. The results from immunofluorescence and biotinylation of cell surface protein showed that V-ATPase ATP6L was conspicuously located in the plasma membrane of human HCC cells. Bafilomycin A1, a specific V-ATPase inhibitor, markedly slowed the intracellular pH (pHi) recovery after acid load in human HCC cells and retarded the growth of human HCC in orthotopic xenograft model. These results demonstrated that V-ATPase is up-regulated in human HCC and involved in the regulation of pHi of human HCC cells. The inhibition of V-ATPase can effectively retard the growth of HCC, indicating that V-ATPase may play an important role in the development and progression of human HCC, and targeting V-ATPase may be a promising therapeutic strategy against human HCC.

[Systemic distribution of prorenin and its receptor].

The (pro)renin receptor was first identified as a 350-amino acid protein with a single transmembrane domain. This receptor binds to prorenin to mediate its dual functions: activation of ERK1/2 independently from angiotensin II generation and induction of full enzymatic activity to initiate angiotensin II-dependent effects. (Pro) renin receptor has recently been shown to undergo intracellular processing, such that it exists in three different molecular forms. These include the full-length (pro)renin receptor, truncated amino-terminal soluble fragment, and carboxy-terminal fragment containing an accessory protein of the vacuolar-type H(+)-ATPase. Their exact distributions and existing molecular forms remain to be determined.

Angiotensin II stimulates H⁺-ATPase activity in intercalated cells from isolated mouse connecting tubules and cortical collecting ducts.

Intercalated cells in the collecting duct system express V-type H(+)-ATPases which participate in acid extrusion, bicarbonate secretion, and chloride absorption depending on the specific subtype. The activity of H(+)-ATPases is regulated by acid-base status and several hormones, including angiotensin II and aldosterone. Angiotensin II stimulates chloride absorption mediated by pendrin in type B intercalated cells and this process is energized by the activity of H(+)-ATPases. Moreover, angiotensin II stimulates bicarbonate secretion by the connecting tubule (CNT) and early cortical collecting duct (CCD). In the present study we examined the effect of angiotensin II (10 nM) on H(+)-ATPase activity and localization in isolated mouse connecting tubules and cortical collecting ducts. Angiotensin II stimulated Na(+)-independent intracellular pH recovery about 2-3 fold, and this was abolished by the specific H(+)-ATPase inhibitor concanamycin. The effect of angiotensin II was mediated through type 1 angiotensin II receptors (AT(1)-receptors) because it could be blocked by saralasin. Stimulation of H(+)-ATPase activity required an intact microtubular network--it was completely inhibited by colchicine. Immunocytochemistry of isolated CNT/CCDs incubated in vitro with angiotensin II suggests enhanced membrane associated staining of H(+)-ATPases in pendrin expressing intercalated cells. In summary, angiotensin II stimulates H(+)-ATPases in CNT/CCD intercalated cells, and may contribute to the regulation of chloride absorption and bicarbonate secretion in this nephron segment.

Modified noninvasive microtest electrophysiological technology for vacuolar H(+) flux detection.

This paper describes a modified noninvasive microtest electrophysiological technology (NMT) for vacuolar H(+) flux detection. In this NMT system, the vacuole isolation procedure and buffer slope were modified, and the measuring errors from small spherical geometry were corrected. The trends in changes of vacuolar H(+) flux (ΔH(+) flux) after ATP or PP(i) supply calculated by NMT were consistent with the activities of V-ATPase and PPase measured by traditional methods. These findings indicate that our modified NMT is an appropriate method for vacuolar H(+) flux detection.

Sarkosyl is a good regeneration reagent for studies on vacuolar-type ATPase subunit interactions in Biacore experiments.

Biacore is widely used for studies on protein-protein interaction in which regeneration is one of the most important steps. Here we introduce the anionic detergent sodium lauroyl sarcosinate (sarkosyl), which works satisfactorily as a regeneration reagent. After regeneration by the mild detergent, the subsequent binding experiment was reproducible without any degradation of the ligand. This regeneration condition can be employed for diverse combinations of ligand-analyte binding interactions and optimized as required.

Prognostic and immunological value of ATP6AP1 in breast cancer: implications for SARS-CoV-2.

Abnormal ATPase H+ Transporting Accessory Protein 1 (ATP6AP1) expression may promote carcinogenesis. We investigated the association of ATP6AP1 with breast cancer (BC) and COVID-19. The Oncomine, Gene Expression Profiling Interactive Analysis, Human Protein Atlas and Kaplan-Meier plotter databases were used to evaluate the expression and prognostic value of ATP6AP1 in BC. ATP6AP1 was upregulated in BC tissues, and higher ATP6AP1 expression was associated with poorer outcomes. Data from the Tumor Immune Estimation Resource, Tumor-Immune System Interaction Database and Kaplan-Meier plotter indicated that ATP6AP1 expression correlated with immune infiltration, and that its prognostic effects in BC depended on tumor-infiltrating immune cell subtype levels. Multiple databases were used to evaluate the association of ATP6AP1 with clinicopathological factors, assess the mutation and methylation of ATP6AP1, and analyze gene co-expression and enrichment. The ATP6AP1 promoter was hypomethylated in BC tissues and differentially methylated between different disease stages and subtypes. Data from the Gene Expression Omnibus indicated that ATP6AP1 levels in certain cell types were reduced after SARS-CoV-2 infections. Ultimately, higher ATP6AP1 expression was associated with a poorer prognosis and with higher or lower infiltration of particular immune cells in BC. BC patients may be particularly susceptible to SARS-CoV-2 infections, which may alter their prognoses.

Analysis of Cry8Ka5-binding proteins from Anthonomus grandis (Coleoptera: Curculionidae) midgut.

Biotech crops expressing Bacillus thuringiensis Cry toxins present a valuable approach for insect control. Cry8Ka5, which is highly toxic to the cotton boll weevil (Anthonomus grandis), was used as a model to study toxin-ligand interactions. Three Cry-binding proteins were detected after toxin overlay assays. Following de novo sequencing, a heat-shock cognate protein and a V-ATPase were identified, whilst a approximately 120 kDa protein remained unknown. Additional Cry8Ka5-binding proteins were visualized by two-dimensional gel electrophoresis ligand blots.

Immunolocalization of IP3R and V-ATPase in Ameloblastomas.

The goal of this study was to investigate the immunolocalization of inositol 1,4,5-trisphosphate receptor (IP3R) and vacuolar ATPase (V-ATPase) in ameloblastomas with special attention to the invasive front. Thirty-seven cases of previously diagnosed formalin-fixed paraffin-embedded (FFPE) human ameloblastoma samples were selected for this study. The samples were grouped according to the predominant histologic pattern and comprised twelve plexiform, eighteen follicular, and seven unicystic ameloblastomas. Of the unicystic variants, six demonstrated purely luminal and intraluminal growth, and one displayed mural extension. One granular cell variant was included in the follicular ameloblastoma group. All specimens were evaluated for IP3R and V-ATPase expression by immunohistochemistry (IHC). IP3R was positive in columnar cells, similar to ameloblasts, and non-peripheral cells in all samples. In the area of tumor protrusion and front of invasion, membranous and cystoplasmic IP3R expression was observed. In contrast, areas adjacent to tumoral protrusion demonstrated only membranous staining patterns. V-ATPase was not expressed in peripheral columnar cells of the unicystic and granular cell variants of ameloblastoma; however, strong staining was present in these cells in plexiform ameloblastomas, follicular ameloblastomas, and areas of mural growth of unicystic ameloblastomas. In areas of tumor protrusion, reactivity for V-ATPase was observed with both membranous and cytoplasmic staining, while other areas showed only membranous V-ATPase. These findings suggest that concomitant immunolocalization of IP3R and V-ATPase, with both cytoplasmic and membranous expression in the peripheral columnar cells, may indicate the invasive potential of ameloblastomas. Furthermore, these results suggest the tumoral spread of ameloblastomas may be correlated with the autophagy process and channelopathy. The expression of these proteins could establish a baseline for future research and provide therapeutic targets for treatment of ameloblastomas.

Francisella tularensis phagosomal escape does not require acidification of the phagosome.

Following uptake, Francisella tularensis enters a phagosome that acquires limited amounts of lysosome-associated membrane glycoproteins and does not acquire cathepsin D or markers of secondary lysosomes. With additional time after uptake, F. tularensis disrupts its phagosomal membrane and escapes into the cytoplasm. To assess the role of phagosome acidification in phagosome escape, we followed acidification using the vital stain LysoTracker red and acquisition of the proton vacuolar ATPase (vATPase) using immunofluorescence within the first 3 h after uptake of live or killed F. tularensis subsp. holarctica live vaccine strain (LVS) by human macrophages. Whereas 90% of the phagosomes containing killed LVS stained intensely for the vATPase and were acidified, only 20 to 30% of phagosomes containing live LVS stained intensely for the vATPase and were acidified. To determine whether transient acidification might be required for phagosome escape, we assessed the impact on phagosome permeabilization of the proton pump inhibitor bafilomycin A. Using electron microscopy and an adenylate cyclase reporter system, we found that bafilomycin A did not prevent phagosomal permeabilization by F. tularensis LVS or virulent type A strains (F. tularensis subsp. tularensis strain Schu S4 and a recent clinical isolate) or by "F. tularensis subsp. novicida," indicating that F. tularensis disrupts its phagosomal membrane by a mechanism that does not require acidification.

The V-ATPase B1-subunit promoter drives expression of Cre recombinase in intercalated cells of the kidney.

The collecting duct of the kidney is composed of two morphologically and physiologically distinct cell types, principal and intercalated cells. To better understand intercalated cell function we generated a transgenic mouse expressing Cre recombinase under the control of a cell type- specific promoter. We used 7 kb of the ATP6V1B1 5' untranslated region (B1 promoter), a gene found in the intercalated cells of the kidney and the male reproductive tract. We first crossed these B1-Cre transgenic mice with the ROSA26-loxP-stop-loxP-yellow fluorescent protein reporter mice to assess the specificity of Cre expression. Immunohistochemistry and confocal fluorescence microscopy showed that Cre is selectively active in all intercalated cells (type A, type B, and non-A/B cells) within the collecting duct and most cells of the connecting segment. About half of the principal cells of the connecting segment also expressed Cre, a pattern also seen in B1-driven enhanced green fluorescent protein transgenic mice. Cre was found to be active in the male reproductive tract and at a low level in limited non-ATP6V1B1 expressing tissues. The B1-Cre transgenic mice are healthy, breed normally, produce regular sized litters, and transmit the transgene in Mendelian fashion. This new cell-specific Cre expressing mouse should prove useful for the study of intercalated cell physiology and development.