Recombinant Mouse V-type proton ATPase subunit G 3 (Atp6v1g3)

What is Atp6v1g3 and where is it predominantly expressed?

Atp6v1g3 is one of three isoforms of the V-ATPase G subunit in the mammalian genome, with the others being Atp6v1g1 (G1) and Atp6v1g2 (G2). While G1 is ubiquitously expressed in various tissues and cells, and G2 shows brain-specific distribution, G3 demonstrates highly specific expression in kidney intercalated cells . The G subunit is a small 13-kDa protein composed of approximately 118 amino acids that forms part of the V-ATPase complex, which is responsible for proton transport across cellular membranes . Expression analysis using the Human Protein Atlas confirms that ATP6V1G3 is predominantly kidney-specific, with minimal expression in other organ systems . Within the kidney, Atp6v1g3 is highly enriched in α-intercalated cells (A-ICs), which are specialized cells in the collecting duct involved in acid-base regulation .

How does Atp6v1g3 differ structurally and functionally from other G subunit isoforms?

The G3 subunit differs from other G isoforms primarily in its highly restricted tissue expression pattern, with G1 being ubiquitously expressed and G2 being primarily neuron-specific . Structurally, the G subunit interacts with the E subunit to form a rod-like structure that connects the V1 catalytic domain and V0 transporting domains of the V-ATPase complex, making it crucial for enzyme activity regulation . The kidney-specific expression of G3 contrasts with the more widespread expression of other V-ATPase subunits such as the B1 subunit (Atp6v1b1), which while enriched in kidney is also found in multiple other organ systems including brain, heart, liver, lung, small intestine, and spleen . This selective tissue distribution suggests specialized physiological roles for G3 in kidney function that cannot be fulfilled by other G isoforms despite their structural similarities.

What is the role of Atp6v1g3 in V-ATPase complex assembly and function?

The G subunit, including G3, plays a critical structural role in the V-ATPase complex by forming part of the peripheral stalks that connect the V1 and V0 domains . These peripheral stalks (composed of subunits C, E, G, and H) attach the A₃B₃ hexamer of the V1 domain to the N-terminal hydrophilic domain of subunit a in V0 . This connection is essential for the rotary mechanism by which V-ATPases operate, where ATP hydrolysis at catalytic sites in V1 drives rotation of the central stalk and attached proteolipid subunits relative to subunit a . The peripheral stalks containing G subunits serve as stators that prevent rotation of subunit a relative to the A₃B₃ hexamer, thus enabling the proton pumping function . As a kidney-specific isoform, G3 likely confers specialized regulatory properties to V-ATPases in intercalated cells, potentially influencing proton transport efficiency, coupling of ATP hydrolysis to proton transport, or regulation of V-ATPase assembly in these specialized cells .

How are Atp6v1g3-Cre mice generated and validated for kidney research?

Atp6v1g3-Cre (G3-Cre) mice are generated using CRISPR/CAS technology to create a model where Cre recombinase expression is controlled by the Atp6v1g3 promoter, enabling intercalated cell-specific genetic manipulation . The process involves designing guide RNAs targeting the Atp6v1g3 locus, followed by embryo injection and screening of founder animals for correct insertion of the Cre recombinase gene . Validation of these mice involves crossing G3-Cre mice with reporter strains such as Tdtomato^flox/flox^ mice to generate G3-Cre^+^Tdt^+^ progeny where Cre-expressing cells can be visualized by Tdtomato fluorescence . Tissue specificity is confirmed by analyzing multiple organs for reporter expression using flow cytometry, which demonstrates that G3-Cre mice show highly kidney-specific expression with minimal or no expression in other organs compared to the previously used B1-Cre mice (based on Atp6v1b1) . Further validation includes RT-PCR analysis of flow-sorted Tdt^+^ cells, confirming enrichment for intercalated cell marker genes .

What techniques are most effective for identifying and isolating Atp6v1g3-expressing cells?

Flow cytometry using G3-Cre^+^Tdt^+^ reporter mice provides an efficient method for identifying and isolating Atp6v1g3-expressing intercalated cells, as the fluorescent reporter enables direct visualization and sorting of these cells . Immunohistochemistry using anti-ATP6V1G3 antibodies can be used to label collecting duct cells, and co-labeling with anti-AQP2 (to mark principal cells) allows distinction between different cell populations, revealing AQP2^-^ATP6V1G3^+^ intercalated cells as well as a unique population of AQP2^+^ATP6V1G3^+^ cells that may represent hybrid or transitional cells . RT-PCR analysis of sorted cells provides confirmation of intercalated cell identity through detection of marker genes specific to these cells . Single-cell RNA sequencing represents an advanced technique to comprehensively characterize Atp6v1g3-expressing cells, revealing subtypes of intercalated cells and identifying co-expressed genes that may have functional significance . These approaches can be complemented by analysis of V-ATPase activity in isolated cells to correlate Atp6v1g3 expression with proton transport function .

What controls should be included when working with Atp6v1g3-Cre mouse models?

When working with Atp6v1g3-Cre mouse models, researchers should include appropriate Cre-negative littermates as controls to account for any phenotypic effects of genetic background or potential insertional effects of the transgene . Comparison with other intercalated cell-targeting Cre models, such as B1-Cre mice, is valuable to distinguish Atp6v1g3-specific effects from general intercalated cell phenotypes and to evaluate the relative specificity of different genetic approaches . Tissue specificity controls should include analysis of multiple organs beyond the kidney to confirm the restricted expression pattern of Atp6v1g3 and exclude off-target Cre activity in other tissues . Co-labeling experiments with established intercalated cell markers (such as V-ATPase B1 or E1 subunits) and markers of other kidney cell types (such as AQP2 for principal cells or calbindin for distal convoluted tubule cells) are essential to validate the specificity of Cre expression within the kidney . Additionally, experiments should include controls to distinguish possible effects of the Cre recombinase itself from the effects of the targeted gene manipulation, particularly when studying subtle phenotypes .

What roles do Atp6v1g3-expressing intercalated cells play in kidney function?

Atp6v1g3-expressing intercalated cells (ICs) play crucial roles in maintaining acid-base homeostasis through their ability to secrete protons via V-ATPase complexes located on their apical membrane . These specialized cells represent a subset of collecting duct cells that regulate urinary pH and contribute to whole-body acid-base balance, with their dysfunction potentially leading to distal renal tubular acidosis . Recent research has revealed that intercalated cells also function in the kidney's innate defense system, suggesting a dual role in both physiological regulation and protection against uropathogens . The importance of these cells is demonstrated in G3-Cre^+^ihCD59^+/+^ mouse models, where depletion of intercalated cells using intermedilysin results in lower blood pH, consistent with a distal renal tubular acidosis phenotype . The specific contribution of the G3 subunit to these functions may involve specialized regulatory properties of the V-ATPase complex in intercalated cells, potentially influencing the efficiency of proton transport or the regulation of V-ATPase assembly and activity in response to physiological demands .

How can researchers experimentally manipulate Atp6v1g3-expressing cells to study their function?

Researchers can use the G3-Cre mouse model crossed with conditional knockout strains to selectively delete genes of interest in Atp6v1g3-expressing intercalated cells, allowing investigation of their function in normal kidney physiology . Cell depletion models, such as the G3-Cre^+^ihCD59^+/+^ mice, enable the selective elimination of intercalated cells through administration of intermedilysin, providing insights into the physiological consequences of intercalated cell loss . These mice develop lower blood pH following intercalated cell depletion, demonstrating the essential role of these cells in acid-base regulation . For functional studies, ex vivo preparations of collecting ducts from G3-Cre reporter mice can be used to measure proton secretion rates and correlate functional capacity with Atp6v1g3 expression levels . Additionally, intercalated cells can be isolated from G3-Cre^+^Tdt^+^ mice using flow cytometry and subjected to various experimental manipulations in vitro, including exposure to different pH conditions or uropathogens, to study their physiological responses and defense mechanisms .

What is known about the relationship between Atp6v1g3 expression and intercalated cell subtypes?

Intercalated cells in the collecting duct are heterogeneous, with distinct subtypes including α-intercalated cells (A-ICs) that secrete protons and β-intercalated cells (B-ICs) that secrete bicarbonate, and Atp6v1g3 expression varies among these subtypes . Studies using single-cell RNA sequencing have revealed that Atp6v1g3 mRNA is highly enriched specifically in α-intercalated cells in both human and mouse kidneys, suggesting a specialized role in this subset of intercalated cells . Immunolabeling of kidney sections reveals AQP2^-^ATP6V1G3^+^ cells, representing classic intercalated cells, as well as AQP2^+^ATP6V1G3^+^ cells, which likely represent hybrid or transitional cells . These hybrid cells are of particular interest as they may represent cells in the process of differentiation or transdifferentiation between principal and intercalated cell phenotypes, with recent studies suggesting that AQP2^+^ cells can give rise to intercalated cells during development or in response to physiological demands . Understanding the significance of differential Atp6v1g3 expression among intercalated cell subtypes may provide insights into the specialized functions of these cells and the regulatory mechanisms controlling their differentiation and adaptation .

How can Atp6v1g3-Cre mice be utilized to study kidney innate immunity mechanisms?

Atp6v1g3-Cre mice offer a powerful tool for investigating the recently discovered role of intercalated cells in kidney innate immunity by enabling selective genetic manipulation of these cells without affecting immune cells in other tissues . This advantage is particularly significant compared to B1-Cre mice, which show expression in multiple tissues including immune cells, potentially confounding interpretations of immune-related phenotypes . Researchers can cross G3-Cre mice with conditional knockout mice targeting specific innate immune genes to determine their function specifically in intercalated cells, avoiding systemic immune effects that might occur with global knockouts . The high kidney specificity of G3-Cre mice allows for the investigation of local immune responses to uropathogens without confounding effects from altered immunity in other organs, providing cleaner experimental systems for studying kidney-specific defense mechanisms . Additionally, these mice can be used to track intercalated cell responses during urinary tract infections through reporter gene expression, potentially revealing dynamic changes in intercalated cell function, number, or distribution during infection that contribute to host defense .

What are the optimal protocols for isolating and characterizing Atp6v1g3-expressing cells?

For isolating Atp6v1g3-expressing intercalated cells, flow cytometry sorting of kidney cell suspensions from G3-Cre^+^Tdt^+^ mice represents an efficient approach that yields cells with high purity, though researchers should be aware that even with this approach, complete purity is not achieved but rather relative enrichment of the target population . Prior to flow cytometry, kidney tissue should be subjected to enzymatic digestion with collagenase and DNase to create single-cell suspensions, followed by filtration through an appropriate mesh to remove cell clumps that could clog the flow cytometer . Characterization of isolated cells should include RT-PCR analysis for intercalated cell marker genes to confirm their identity and assess the degree of enrichment achieved by the isolation procedure . Immunofluorescence co-labeling with markers such as AQP2 (for principal cells) and V-ATPase subunits like B1 or E1 provides additional validation of cell identity and can reveal hybrid cell populations that express markers of multiple cell types . For comprehensive characterization, single-cell RNA sequencing of isolated cells offers an unbiased approach to identify gene expression patterns, revealing heterogeneity within the intercalated cell population and potentially identifying previously unrecognized cell subtypes .

What are the key considerations for experimental design when using Atp6v1g3-Cre mice for kidney research?

When designing experiments with Atp6v1g3-Cre mice, researchers should first validate Cre expression patterns in their specific colony through reporter gene studies, as expression patterns can vary between generations or breeding environments . Careful selection of control groups is essential, including both Cre-negative littermates and, when possible, comparison with other intercalated cell-targeting Cre lines such as B1-Cre to distinguish G3-specific from general intercalated cell effects . Researchers should be aware of the potential presence of hybrid cells expressing both AQP2 and Atp6v1g3 (approximately 5% of cells), which might confound interpretations if effects are observed in principal cells; co-labeling experiments can help identify these hybrid populations . For phenotypic analyses, comprehensive assessment of acid-base parameters should be included (such as blood pH, bicarbonate levels, and urinary pH) even when studying other aspects of intercalated cell function, as acid-base disturbances might indirectly affect other cellular processes . Additionally, researchers should consider potential differences between sexes, as some kidney functions and V-ATPase expression patterns show sexual dimorphism, necessitating the inclusion of both male and female animals with sufficient sample sizes to detect sex-specific effects .