Carbonic anhydrases (CAs) catalyze a reaction fundamental for life: the bidirectional conversion of carbon dioxide (CO
) and water (H
O) into bicarbonate (HCO
) and protons (H
). These enzymes impact ...numerous physiological processes that occur within and across the many compartments in the body. Within compartments, CAs promote rapid H
buffering and thus the stability of pH-sensitive processes. Between compartments, CAs promote movements of H
, CO
, HCO
, and related species. This traffic is central to respiration, digestion, and whole-body/cellular pH regulation. Here, we focus on the role of mathematical modeling in understanding how CA enhances buffering as well as gradients that drive fluxes of CO
and other solutes (facilitated diffusion). We also examine urinary acid secretion and the carriage of CO
by the respiratory system. We propose that the broad physiological impact of CAs stem from three fundamental actions: promoting H
buffering, enhancing H
exchange between buffer systems, and facilitating diffusion. Mathematical modeling can be a powerful tool for: (1) clarifying the complex interdependencies among reaction, diffusion, and protein-mediated components of physiological processes; (2) formulating hypotheses and making predictions to be tested in wet-lab experiments; and (3) inferring data that are impossible to measure.
The mammalian Slc4 (Solute carrier 4) family of transporters is a functionally diverse group of 10 multi-spanning membrane proteins that includes three Cl-HCO3 exchangers (AE1-3), five Na(+)-coupled ...HCO3(-) transporters (NCBTs), and two other unusual members (AE4, BTR1). In this review, we mainly focus on the five mammalian NCBTs-NBCe1, NBCe2, NBCn1, NDCBE, and NBCn2. Each plays a specialized role in maintaining intracellular pH and, by contributing to the movement of HCO3(-) across epithelia, in maintaining whole-body pH and otherwise contributing to epithelial transport. Disruptions involving NCBT genes are linked to blindness, deafness, proximal renal tubular acidosis, mental retardation, and epilepsy. We also review AE1-3, AE4, and BTR1, addressing their relevance to the study of NCBTs. This review draws together recent advances in our understanding of the phylogenetic origins and physiological relevance of NCBTs and their progenitors. Underlying these advances is progress in such diverse disciplines as physiology, molecular biology, genetics, immunocytochemistry, proteomics, and structural biology. This review highlights the key similarities and differences between individual NCBTs and the genes that encode them and also clarifies the sometimes confusing NCBT nomenclature.
The SLC4 family of bicarbonate (HCO3-) transporters Romero, Michael F.; Chen, An-Ping; Parker, Mark D. ...
Molecular aspects of medicine,
April-June 2013, 2013 Apr-Jun, 2013-4-00, 20130401, Letnik:
34, Številka:
2-3
Journal Article
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The SLC4 family consists of 10 genes (SLC4A1–5; SLC4A7–11). All encode integral membrane proteins with very similar hydropathy plots—consistent with 10–14 transmembrane segments. Nine SLC4 members ...encode proteins that transport HCO3- (or a related species, such as CO32-) across the plasma membrane. Functionally, eight of these proteins fall into two major groups: three Cl–HCO3 exchangers (AE1–3) and five Na+-coupled HCO3- transporters (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE). Two of the Na+-coupled transporters (NBCe1, NBCe2) are electrogenic; the other three Na+-coupled HCO3- transporters and all three AEs are electroneutral. In addition, two other SLC4 members (AE4, SLC4A9 and BTR1, SLC4A11) do not yet have a firmly established function. Most, though not all, SLC4 members are functionally inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS). SLC4 proteins play important roles many modes of acid–base homeostasis: the carriage of CO2 by erythrocytes, the transport of H+ or HCO3- by several epithelia, as well as the regulation of cell volume and intracellular pH.
One of the major tasks of the renal proximal tubule is to secrete acid into the tubule lumen, thereby reabsorbing approximately 80% of the filtered HCO3- as well as generating new HCO3- for ...regulating blood pH. This review summarizes the cellular and molecular events that underlie four major processes in HCO3- reabsorption. The first is CO2 entry across the apical membrane, which in large part occurs via a gas channel (aquaporin 1) and acidifies the cell. The second process is apical H+ secretion via Na-H exchange and H+ pumping, processes that can be studied using the NH4+ prepulse technique. The third process is the basolateral exit of HCO3- via the electrogenic Na/HCO3 co-transporter, which is the subject of at least 10 mutations that cause severe proximal renal tubule acidosis in humans. The final process is the regulation of overall HCO3- reabsorption by CO2 and HCO3- sensors at the basolateral membrane. Together, these processes ensure that the proximal tubule responds appropriately to acute acid-base disturbances and thereby contributes to the regulation of blood pH.
Aquaporin-4 (AQP4) plays an important role in regulating water exchange across the blood-brain barrier (BBB) and brain-cerebrospinal fluid interface. Studies on AQP-4 knockout mice (AQP4-KO) have ...reported considerable protection from brain edema induced by acute water intoxication and ischemic stroke, identifying AQP4 as a potential target for therapeutic interventions. However, the long-term effects of chronic AQP4 suppression are yet to be elucidated. In the current study, we evaluated the physiological and structural changes in adult AQP4-KO mice using magnetic resonance imaging (MRI) and immunohistochemical analysis. Water exchange across BBB was assessed by tracking an intravenous bolus injection of oxygen-17 (17O) water (H217O) using 17O-MRI. Cerebral blood flow (CBF) was quantified using arterial spin-labeling (ASL) MRI. Capillary density was determined by immunohistochemical staining for glucose transporter-1 (GLUT1). Compared to wildtype control mice, AQP4-KO mice showed a significant reduction in peak and steady-state H217O uptake despite unaltered CBF. Interestingly, a 22% increase in cortical capillary density was observed in AQP4-KO mice. These results suggest that increased cerebral vascularization may be an adaptive response to chronic reduction in water exchange across BBB in AQP4-KO mice.
Sharpey‐Schafer Lecture: Gas channels Boron, Walter F.
Experimental physiology,
December 2010, Letnik:
95, Številka:
12
Journal Article, Presentation
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The traditional dogma has been that all gases diffuse through all membranes simply by dissolving in the lipid phase of the membrane. Although this mechanism may explain how most gases move through ...most membranes, it is now clear that some membranes have no demonstrable gas permeability, and that at least two families of membrane proteins, the aquaporins (AQPs) and the Rhesus (Rh) proteins, can each serve as pathways for the diffusion of both CO2 and NH3. The knockout of RhCG in the renal collecting duct leads to the predicted consequences in acid–base physiology, providing a clear‐cut role for at least one gas channel in the normal physiology of mammals. In our laboratory, we have found that surface‐pH (pHS) transients provide a sensitive approach for detecting CO2 and NH3 movement across the cell membranes of Xenopus oocytes. Using this approach, we have found that each tested AQP and Rh protein has its own characteristic CO2/NH3 permeability ratio, which provides the first demonstration of gas selectivity by a channel. Our preliminary AQP1 data suggest that all the NH3 and less than half of the CO2 move along with H2O through the four monomeric aquapores. The majority of CO2 takes an alternative route through AQP1, possibly the central pore at the four‐fold axis of symmetry. Preliminary data with two Rh proteins, bacterial AmtB and human erythroid RhAG, suggest a similar story, with all the NH3 moving through the three monomeric NH3 pores and the CO2 taking a separate route, perhaps the central pore at the three‐fold axis of symmetry. The movement of different gases via different pathways is likely to underlie the gas selectivity that these channels exhibit.
Mathematical modeling of acid-base physiology Occhipinti, Rossana; Boron, Walter F.
Progress in Biophysics and Molecular Biology/Progress in biophysics and molecular biology,
January 2015, 2015-Jan, 2015-01-00, 20150101, Letnik:
117, Številka:
1
Journal Article
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pH is one of the most important parameters in life, influencing virtually every biological process at the cellular, tissue, and whole-body level. Thus, for cells, it is critical to regulate ...intracellular pH (pHi) and, for multicellular organisms, to regulate extracellular pH (pHo). pHi regulation depends on the opposing actions of plasma-membrane transporters that tend to increase pHi, and others that tend to decrease pHi. In addition, passive fluxes of uncharged species (e.g., CO2, NH3) and charged species (e.g., HCO3−, NH4+) perturb pHi. These movements not only influence one another, but also perturb the equilibria of a multitude of intracellular and extracellular buffers. Thus, even at the level of a single cell, perturbations in acid-base reactions, diffusion, and transport are so complex that it is impossible to understand them without a quantitative model. Here we summarize some mathematical models developed to shed light onto the complex interconnected events triggered by acids-base movements. We then describe a mathematical model of a spherical cells—which to our knowledge is the first one capable of handling a multitude of buffer reactions—that our team has recently developed to simulate changes in pHi and pHo caused by movements of acid-base equivalents across the plasma membrane of a Xenopus oocyte. Finally, we extend our work to a consideration of the effects of simultaneous CO2 and HCO3− influx into a cell, and envision how future models might extend to other cell types (e.g., erythrocytes) or tissues (e.g., renal proximal-tubule epithelium) important for whole-body pH homeostasis.
Dark respiration causes an increase in leaf CO2 concentration (Ci), and the continuing increases in atmospheric CO2 further increases Ci. Elevated leaf CO2 concentration causes stomatal pores to ...close. Here, we demonstrate that high intracellular CO2/HCO3
− enhances currents mediated by the Arabidopsis thaliana guard cell S-type anion channel SLAC1 upon coexpression of any one of the Arabidopsis protein kinases OST1, CPK6, or CPK23 in Xenopus laevis oocytes. Split-ubiquitin screening identified the PIP2;1 aquaporin as an interactor of the βCA4 carbonic anhydrase, which was confirmed in split luciferase, bimolecular fluorescence complementation, and coimmunoprecipitation experiments. PIP2;1 exhibited CO2 permeability. Mutation of PIP2;1 in planta alone was insufficient to impair CO2- and abscisic acid-induced stomatal closing, likely due to redundancy. Interestingly, coexpression of βCA4 and PIP2;1 with OST1-SLAC1 or CPK6/23-SLAC1 in oocytes enabled extracellular CO2 enhancement of SLAC1 anion channel activity. An inactive PIP2;1 point mutation was identified that abrogated water and CO2 permeability and extracellular CO2 regulation of SLAC1 activity. These findings identify the CO2-permeable PIP2;1 as key interactor of βCA4 and demonstrate functional reconstitution of extracellular CO2 signaling to ion channel regulation upon coexpression of PIP2;1, βCA4, SLAC1, and protein kinases. These data further implicate SLAC1 as a bicarbonate-responsive protein contributing to CO2 regulation of S-type anion channels.
Carbonic anhydrases (CAs), because they catalyze the interconversion of carbon dioxide (CO
) and water into bicarbonate (HCO
) and protons (H
), thereby influencing pH, are near the core of virtually ...all physiological processes in the body. In the kidneys, soluble and membrane-associated CAs and their synergy with acid-base transporters play important roles in urinary acid secretion, the largest component of which is the reabsorption of HCO
in specific nephron segments. Among these transporters are the Na
-coupled HCO
transporters (NCBTs) and the Cl
-HCO
exchangers (AEs)-members of the "solute-linked carrier" 4 (SLC4) family. All of these transporters have traditionally been regarded as "HCO
" transporters. However, recently our group has demonstrated that two of the NCBTs carry CO
rather than HCO
and has hypothesized that all NCBTs follow suit. In this review, we examine current knowledge on the role of CAs and "HCO
" transporters of the SLC4 family in renal acid-base physiology and discuss how our recent findings impact renal acid secretion, including HCO
reabsorption. Traditionally, investigators have associated CAs with producing or consuming solutes (CO
, HCO
, and H
) and thus ensuring their efficient transport across cell membranes. In the case of CO
transport by NCBTs, however, we hypothesize that the role of membrane-associated CAs is not the appreciable production or consumption of substrates but the minimization of pH changes in nanodomains near the membrane.
The water channel aquaporin 1 (AQP1) and certain Rh-family members are permeable to CO₂ and NH₃. Here, we use changes in surface pH (pHS) to assess relative CO₂ vs. NH₃ permeability of Xenopus ...oocytes expressing members of the AQP or Rh family. Exposed to CO₂ or NH₃, AQP1 oocytes exhibit a greater maximal magnitude of pHS change (ΔpHS) compared with day-matched controls injected with H₂O or with RNA encoding SGLT1, NKCC2, or PepT1. With CO₂, AQP1 oocytes also have faster time constants for pHS relaxation (τpHs). Thus, AQP1, but not the other proteins, conduct CO₂ and NH₃. Oocytes expressing rat AQP4, rat AQP5, human RhAG, or the bacterial Rh homolog AmtB also exhibit greater ΔpHS(CO₂) and faster τpHs compared with controls. Oocytes expressing AmtB and RhAG, but not AQP4 or AQP5, exhibit greater ΔpHS(NH₃) values. Only AQPs exhibited significant osmotic water permeability (Pf). We computed channel-dependent (*) ΔpHS or Pf by subtracting values for H₂O oocytes from those of channel-expressing oocytes. For the ratio ΔpHS(CO₂)*/PFormula: see text*, the sequence was AQP5 > AQP1 congruent with AQP4. For ΔpHS(CO₂)*/ΔpHS(NH₃)*, the sequence was AQP4 congruent with AQP5 > AQP1 > AmtB > RhAG. Thus, each channel exhibits a characteristic ratio for indices of CO₂ vs. NH₃ permeability, demonstrating that, like ion channels, gas channels can exhibit selectivity.
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