Morris F. Manolson

Chronic Extracellular Acidosis Induces Plasmalemmal Vacuolar Type H+ ATPase Activity in Osteoclasts

Proton extrusion into an extracellular resorption compartment is an essential component of bone degradation by osteoclasts. Chronic metabolic acidosis is known to induce negative calcium balance and bone loss by stimulating osteoclastic bone resorption, but the underlying mechanism is not known. The present studies were undertaken to evaluate whether chronic acidosis affects proton extrusion mechanisms in osteoclasts cultured on glass coverslips. Acidosis, mimicked experimentally by maintaining the cells at extracellular pH 6.5, rapidly lowered intracellular pH to 6.8. However, after 2 hours, a proportion of cells demonstrated the capacity to restore intracellular pH to near normal levels. To define the mechanism responsible for this recovery, the activity of individual H+ transport pathways was analyzed. We found that chronic acid treatment for up to 6 h did not significantly affect the cellular buffering power or Na+/H+ antiport activity. In contrast, chronic acidosis activated vacuolar H+ pumps in the osteoclasts. Although only ∼5% of the control cells displayed proton pump activity, about 40% of cells kept at extracellular pH 6.5 for 4-6 h were able to recover from the acute acid load by means of bafilomycin A1-sensitive proton extrusion. Conversely, the H+-selective conductance recently described in the plasma membrane of osteoclasts was clearly inhibited in the cells exposed to chronic acidosis. Following acid treatment, the activation threshold of the H+ conductance was shifted to more positive potentials, and the current density was significantly reduced. Considered together, these results suggest that induction of plasmalemmal vacuolar type ATPase activity by chronic acidosis, generated either systemically due to metabolic disease or locally at sites of inflammation, is likely to stimulate osteoclastic bone resorption and thus to promote bone loss.

Irreversible inhibition of H+-ATPase of higher plant tonoplast by chaotropic anions: evidence for peripheral location of nucleotide-binding subunits

Abstract The H+-translocating ATPase of tonoplast vesicles from storage root of Beta vulgaris L. is irreversibly inhibited by a 30 min treatment with the chaotropic anions SCN−, ClO4−, I− or NO3− in the 0.25–1.5 M concentration range. The inhibitory potencies of the anions follow the Hofmeister series ( SCN − > ClO 4 − > I − > NO 3 − > CH 3 COO − ; SO 4 2− = 0 ). The H+-translocating inorganic pyrophosphatase of the same membrane is, by contrast, unaffected by chaotrope concentrations which completely abolish H+-ATPase activity. Inhibition of the ATPase is associated with the removal of two polypeptides of 67 and 57 kDa from the membrane, concomitant with their appearance in the supernatant. The chaotrope-dissociated 67 and 57 kDa polypeptides comigrate with the major subunits of the partially purified ATPase upon SDS-polyacrylamide gel electrophoresis and cross-react with antibody raised to the nucleotide-binding subunits of the enzyme. Since the 16 kDa [14C]DCCD-binding proteolipid of the ATPase remains associated with the membrane after treatment with chaotrope, it is concluded that chaotropic anions inhibit the enzyme by specific detachment of the nucleotide-binding subunits. The tonoplast ATPase of Beta is therefore deduced to have structure/function partitioning analogous to the F0F1 H+-ATPase of energy-coupling membranes.

Inhibition of osteoclast bone resorption by disrupting vacuolar H+-ATPase a3-B2 subunit interaction.

Vacuolar H+-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC50 of ∼1.2 μm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis.

Control of Excitatory Synaptic Transmission by C-terminal Src Kinase

The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-d-aspartate (NMDA) receptordependent accumulation of intracellular Ca2+, followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca2+ into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus.

Protein kinase C activation accelerates proton extrusion by vacuolar-type H+-ATPases in murine peritoneal macrophages

The role of protein kinase C in the regulation of vacuolar‐type H+‐ATPase (V‐ATPase) activity was studied in thioglycolate‐elicited mouse peritoneal macrophages. Acid‐loaded macrophages suspended in a Na+‐ and HCO− 3‐free K+‐medium containing Zn2+ , a H+‐conductance blocker, exhibited an initial intracellular pH recovery rate of 0.33 ± 0.04 pH/min (n = 9). Pretreatment with 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) or mezerein for as little as 3 min induced a marked (82%) increase in the initial pH recovery rate. Stimulation was prevented by the V‐ATPase inhibitor, bafilomycin A1 (200 nM) indicating that the effect of the protein kinase C agonists was via augmentation of proton pump activity. The protein kinase C inhibitor, staurosporine (100 nM) completely blocked the stimulatory effects of TPA and mezerein, suggesting involvement of protein kinase C. In keeping with this notion, the inactive analogue of TPA, 4‐phorbol didecanoate did not stimulate recovery from an acid load. Extracellular pH determinations revealed that the observed increase in cytosolic pH recovery rate by the protein kinase C agonists was due to increased extrusion of protons from the cells, likely through V‐ATPases located in the plasma membrane. Considered together, these data demonstrate regulation of plasmalemmal V‐ATPase‐mediated proton extrusion by protein kinase C.

Osteopetrosis mutation R444L causes endoplasmic reticulum retention and misprocessing of vacuolar H+-ATPase a3 subunit.

Background: The human V-ATPase a3 subunit mutation, R444L, causes infantile malignant osteopetrosis. Results: In mouse, the R444L equivalent, R445L, causes endoplasmic reticulum retention, misprocessing, and defective trafficking of a3 to the plasma membrane. Conclusion: Arginine 444/445 plays a critical role in mammalian a3 folding, or stability. Significance: R444L a3 infantile malignant osteopetrosis is a protein folding disease that may be amenable to protein rescue therapy. Osteopetrosis is a genetic bone disease characterized by increased bone density and fragility. The R444L missense mutation in the human V-ATPase a3 subunit (TCIRG1) is one of several known mutations in a3 and other proteins that can cause this disease. The autosomal recessive R444L mutation results in a particularly malignant form of infantile osteopetrosis that is lethal in infancy, or early childhood. We have studied this mutation using the pMSCV retroviral vector system to integrate the cDNA construct for green fluorescent protein (GFP)-fused a3R445L mutant protein into the RAW 264.7 mouse osteoclast differentiation model. In comparison with wild-type a3, the mutant glycoprotein localized to the ER instead of lysosomes and its oligosaccharide moiety was misprocessed, suggesting inability of the core-glycosylated glycoprotein to traffic to the Golgi. Reduced steady-state expression of the mutant protein, in comparison with wild type, suggested that the former was being degraded, likely through the endoplasmic reticulum-associated degradation pathway. In differentiated osteoclasts, a3R445L was found to degrade at an increased rate over the course of osteoclastogenesis. Limited proteolysis studies suggested that the R445L mutation alters mouse a3 protein conformation. Together, these data suggest that Arg-445 plays a role in protein folding, or stability, and that infantile malignant osteopetrosis caused by the R444L mutation in the human V-ATPase a3 subunit is another member of the growing class of protein folding diseases. This may have implications for early-intervention treatment, using protein rescue strategies.

V-ATPase Subunit Interactions: The Long Road to Therapeutic Targeting

Over the last three decades, V-ATPases have emerged from the obscurity of poorly understood membrane proton transport phenomena to being recognized as ubiquitous proton pumps that underlie vital cellular processes in all eukaryotic and many prokaryotic cells. These exquisitely complex molecular motors also engage in diverse specialized roles contributing to development, tissue function and pH homeostasis within complex organisms. Increasingly, mutations and misappropriation of V-ATPase function have been linked to diseases, ranging from sclerosing bone pathologies and renal tubular acidosis to bone-loss disorders and cancer metastasis. Much remains to be learned about the details of V-ATPase cell and molecular biology; nevertheless, interest in V-ATPases as potential therapeutic targets has burgeoned in recent years. In this review, we present a history of our involvement and contributions to the understanding of V-ATPase structure and function and our nascent and ongoing contributions to translating the knowledge gained from basic research on the nature of V-ATPases into tools for drug discovery. We focus here primarily on the treatment of bone-loss pathologies, like osteoporosis, and present proof-of-concept for a drug screening strategy based on targeting a3-B2 subunit interactions within the V-ATPase complex.