Cellular mechanisms for electrolyte and water homeostasis Objectives: Principle of Na + and water transport Amiloride sensitive Na + Channels CFTR and transepithelial Cl transport Aquaporins in water homeostasis TRP Channels and Vitamin D dependent Ca ++ transport 1 Principle of Na + and water transport in epithelial cells Na + and Cl are the major ions in the interstitial fluid, and they have opposite charges. When Na + or Cl is transported across plasma membranes, the shift in electrical charges tends to bring the other with it. Such a movement can be produced by membrane transporters and ion channels. The Na + and Cl movements determine the water movement. 2 1
Amiloride sensitive Na + Channels in Epithelial Cells 3 Expression of renal Na + transporters 4 2
Model of the pore region of the KcsA K+ channel and of ENaC. A: the model is based on the crystal structure of the KcsA K+ channel and shows the pore region. Two of the four subunits are shown. The second transmembrane domains, the "inner helices," are shown in black; the pore helices are in gray; and the selectivity filter GYG is indicated. B: the model of the ENaC pore is based on functional data of wild type and mutant ENaC. The pre M2 segments and the second putative transmembrane helices (M2) of two of the four ENaC subunits are shown. Amino acid numbering of the subunit is indicated. In contrast to KcsA subunits which contain only ~30 residues between the two transmembrane segments M1 and M2, the extracellular portion of ENaC subunits represents >50% of the subunit mass and extends much farther outward than shown in the figure. The outer vestibule is lined by the pre M2 segments with the site where amiloride binds to S583 and the corresponding Gly residues. The vestibule narrows down to the selectivity filter formed by G587, G529, S541, and the ring of Ser residues. In this model the M2 domain forms the intracellular part of the pore, in analogy to K+ channels. 5 ENaC regulation PDK, phosphoinositide-dependent kinase; N4-2, Nedd4-2. 6 3
Aldosterone / ADH Stimulates transcription, Enhances protein surface expression, and Activates the channel through the activation of camp dependent protein kinase (PKA). 7 Functions The epithelial amiloride sensitive Na + channel plays a role in Na + reabsorption by the epithelial cells in the distal nephrons. ENaC functional activity and surface expression are subject to extensive physiological regulations. 8 4
The Na + reabsorption affects: Salt balance, Osmolarity, Blood volume and Blood pressure. 9 CFTR and transepithelial Cl transport Cystic fibrosis is caused by mutations in an ATP/ADP regulated Cl channel known as cystic fibrosis transmembraneconductance regulator (CFTR). The CFTR is found in the apical membrane of exocrine epithelial cells. 10 5
Cl channels perform various physiologic tasks Membrane potential maintenance, Acid base balance, Signal transduction, Cell volume regulation, Transepithelial transport and Acid secretion. 11 Single channel currents recorded from an excised, inside out patch. Bath (cytosolic) solution contained indicated concentration of ATP. Closed state is indicated; downward deflections represent channel opening. Holding voltage was 120 mv. [Adapted from Winter et al. (145).] 12 6
Model showing proposed domain structure of cystic fibrosis transmembrane conductance regulator (CFTR). MSD, membrane spanning domain; NBD, nucleotide binding domain; R, regulatory domain; PKA, campdependent protein kinase. 13 CFTR is involved in: Cl transportation, Conducting Cl currents, Positive regulation of CLC channels, Positive regulation of a Ca ++ dependent Cl channels, and Negative regulation of the amiloride sensitive Na + channels. 14 7
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17 18 9
Cystic fibrosis CF is a systemic disease affecting multiple organs and tissues. The molecular basis of CF is the dysfunction of CFTR. The wide existence of CF cases suggests that early humans may have benefited from CFTR mutations, as a decrease in epithelial Cl - transport can reduce liquid secretion such as during diarrhea. 19 Aquaporins, the facilitative water transporters Aquaporins are a group of membrane proteins responsible for water molecules to pass across plasma membranes. Functional water channels are made of four aquaporins. Each subunit has a water permeable pore. These molecules are sensitive to heavy metal such as Hg ++ and Cd ++. 20 10
Structural features conserved throughout the aquaporin family. (a) The common fold. Half helices formed by loops B and E are colored yellow, and water molecules inside the water conducting channel are colored orange. (b) The conserved tetrameric arrangement. (c) Stereo model of the water conducting channel, showing electron density for seven water molecules, the conserved ar/r constriction region and the NPA signature motif. The X ray structure of closed SoPIP2;1 is used to illustrate this fold and also shows the position of Leu197, which gates the channel. 21 Snapshot from MD simulation revealing the orientation of the hydrogen bonded water network that precludes proton conduction in GlpF. The opposite orientations of water molecules in the two halves of the channel start from the central water molecule opposite the NPA motifs (residues 68 70 and 203 205 shown in green with the side chains of Asn68 and Asn203 displayed in licorice representation), and are stabilized by the electrostatic fields generated by the M3 and M7 helices (shown in blue), and by hydrogen bonds to the carbonyl groups in the non helical parts of two halfmembrane spanning repeats (shown in gray). The exposed carbonyl oxygens of residues 65 66 and 198 200 are displayed as red spheres. Residues Arg206, Phe200, and Trp48 constituting the selectivity filter appear in light blue. 22 11
Movements of Met176 in AQP0 gating 23 Regulation of AQP2 trafficking and expression in collecting duct principal cells. ADH acts on V2 receptors (V2R) in the basolateral plasma membrane (A and B). A: through the GTPbinding protein G S adenylyl cyclase (AC) is activated, producing camp and activating PKA. PKA phosphorylates AQP2 in intracellular vesicles and possible other cytosolic or membrane proteins. Specifically, camp participates in the long term regulation of AQP2 by increasing the levels of the catalytic subunit of PKA in the nuclei, which is thought to phosphorylate transcription factors such as CREB P (camp responsive element binding protein) and c Jun/c Fos. Binding of these factor may increase gene transcription of AQP2 resulting in synthesis of AQP2 protein which in turn enters the regulated trafficking system. In parallel, AQP3 synthesis and trafficking to the basolateral membrane takes place. B: AQP2 is excreted into urine or recycled from the apical plasma membrane. 24 12
AQP Regulation Transcription Expression targeting to different membranes Direct gating of the AQPs in situ. Vasopressin (antidiuretic hormone, ADH) regulates the water permeability of the kidney collecting duct via AQP and ENaC. 25 TRP Channels and Vitamin D dependent Ca ++ transport Transient receptor potential (TRP) channels were first discovered and characterized in Drosophila. These channels are called transient receptor potential because a mutation in the gene encoding the channel results in a transient depolarization of photoreceptor cells in Drosophila. Since the discovery of the TRP gene in Drosophila, many TRP homologues have been cloned from mammals. 26 13
(a) Transient receptor potential (TRP) ion channel family subgroup. (Only main members are shown.) Short, osm 9 related and long TRP channel nomenclature is shown [23] and the new proposed nomenclature is shown in parentheses on the right [24 and 25] (see Table 2 in the main text for details). Note that human STRPC2 is a pseudogene and is not depicted in this diagram. (b) The predicted protein structure of TRP proteins. Predicted membrane topology of monomeric TRP polypeptides [26] is shown with the location of putative pore region (left) and functional channel tetrameric subunit structure (right). (c) Regulatory and protein interaction sites on TRP channel proteins. The general characteristics, such as N terminal location of ankyrin repeats in STRPCs and OTRPCs (orange circles) and the proline rich regions of STRPCs and LTRPCs (green circles), of the STRPC, LTRPC and OTRPC subfamily are shown. The presence of some predicted protein kinase A (PKA) and C (PKC) and phosphatidylinositide 3 kinase (PI3K) SH2 recognition domains (YXXM motifs) [58] are also depicted (results of GCG computational analysis of amino acid sequences from accession numbers in Table 2). The presence of putative PDZ interacting motifs is shown for STRPC4 and 5 [24]. Note that this is not a complete and comprehensive list of putative regulatory sites and merely highlights the presence of such regions in TRP channel proteins. Abbreviations: CaM, calmodulin; IP3R, inositol (1,4,5) trisphosphate receptor; LTRPC, long transient receptor potential channel; MHCK/eEF2?, myosin heavy chain kinase/eukaryotic elongation factor 2 family of protein kinases; OTRPC, osm 9 like transient receptor potential channel; PDZ interacting motifs, peptide binding domains that are important for the scaffolding and organization of membrane proteins and named after the proteins in which these sequences were originally identified (i.e. SD95, iscs large, ona occludens 1) [24]; STRPC, short transient receptor potential channel; VR1, vanilloid receptor 1; VRL 1, vanilloid receptor like 1. 27 Pairwise similarity phylogenetic tree of the TRP family. Two distinct subgroups can be identified by their phylogenetic relationship. The top group (except for Drosophila TRP and TRPL) shows murine members of the "TRP homolog" (TRPC) group. The bottom group includes several members of the "TRP related" group. The scale represents evolutionary distance calculated by Clustal analysis. 28 14
Luminal Ca 2+ influx is mediated by TRPV5 in the renal distal convoluted and connecting tubules and TRPV6 in the duodenum. 29 Luminal Mg 2+ influx is mediated by TRPM6 in the renal distal convoluted tubules and the intestine. 30 15
31 Calbindin Molecular model for intestinal calcium absorption. Paracellular calcium absorption is largely a passive mechanism driven by luminal calcium concentration and the integrity of intercellular tight junction. The transcellular pathway involves the epithelial calcium entry through specialised calcium channels (ECaC2 and to a much lesser extent ECaC1). These channels seem to be constitutively in the open modulus and influx can therefore be regulated by the number of channels. These channels, however, are highly dependent on intracellular free calcium concentration and to remain in the open state requires the constant buffering of entering calcium by intracellular calcium binding proteins. Calbindin D9K is the major intracellular calcium binding protein in the mammalian intestinal cell but the contribution of other proteins (CaBP D28K, calmodulin, etc.) is unknown. The molecular mechanism involved in the transfer of calcium between the different proteins is also not fully explored (direct protein protein interaction or other helper proteins involved?). The final extrusion of calcium toward the plasma requires ATP driven mechanisms, whereby PMCA1b has a much more important role than NCX. A not fully explored mechanism might involve the calbindin mediated direct uptake into the circulation; however such mechanism would not allow the massive amount of calcium transport (>300 mg/day). 32 16
Coordinated model of the mechanism by which CaT1 and calbindin D9k increase calcium entry in response to 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]. In the absence of 1,25(OH)2D3 (left), ECaC2 (CaT1) and calbindin D9k levels are low. When CaT1 is open, calcium entry will increase the local calcium concentration beneath the membrane. Increased local calcium concentration results in calmodulin binding to CaT1 and shutting off the channel. In the presence of 1,25(OH)2D3 (right), however, the levels of both CaT1 and calbindin D9k are increased. Although the amount of calcium entry is increased, the local free calcium level is kept low as calbindin D9k binds and buffers calcium. Thus calcium flow through the apical membrane is increased in the presence of 1,25(OH)2D3 because the number of CaT1 channels increases and the feedback inhibition by intracellular calcium is released by the calcium buffering effect of calbindin D9k. 33 Ca 2+ absorption and absorption Ca 2+ absorption and reabsorption rely on ECaC channels and calbindin in epithelial cells. The ECaC channels are inhibited by Ca 2+ calmodulin. Ca 2+ binding to calmodulin is inhibited when clabindin concentration is high, releasing the ECaC channel inhibition. Both levels of ECaC channel and calbindin are regulated by 1,25 dihydroxyvitamin D3. 34 17
ECaC Functions The epithelial Ca ++ channels (ECaCs) are primarily expressed in Ca ++ transporting epithelia and represent a new family of Ca ++ channels that belong to the superfamily of the TRP channels. Two members, namely ECaC1 and ECaC2, have been identified from kidney and intestine, respectively. These channels are the prime target for hormonal control of active Ca ++ flux from the urine space or intestinal lumen to the blood compartment. 35 18