Which 3 transporters can let ions into the cell?

Alveolar Fluid Clearance and Ion Transport

An intact barrier is also required to keep the alveolus free of fluid. The close apposition between the alveolar epithelium and the vascular endothelium, in addition to facilitating gas exchange, creates a tight barrier restricting passive movement of liquid, ions, and proteins from the interstitial and vascular spaces, thus assisting in maintaining the relatively dry condition of the alveolar air spaces.265 Of note, the alveolar epithelium, rather than the endothelium, is believed to be the chief permeability barrier to small, water-soluble molecules.266,267 Therefore, directed transport of ions, fluid, and proteins across this epithelial layer is essential for maintenance of normal gas exchange and for resolution of both cardiogenic and noncardiogenic pulmonary edema. This transport is made possible via a combination of active and passive transport, mediated by both ion channels and ion pumps.268 Channels, when open, allow the passive diffusion of ions rapidly down electrical and concentration gradients, which results in the generation of transmembrane electric currents. In contrast, pumps actively transport ions against the electrochemical gradient in an energy-dependent fashion. Ion transporters and other membrane proteins asymmetrically distributed on apical and basolateral surfaces coordinately regulate the directional transport properties of the alveolar epithelium (seeFig. 3.8).269,270

For many years, differences in hydrostatic and protein osmotic pressures (Starling forces) were thought to account for the removal of excess fluid from the air spaces of the lung.270,271 Evidence for the contribution of active ion transport toalveolar fluid clearance (AFC) came from extensive in vivo and in vitro experimental studies.270,272–274 Intratracheally administered fluid is cleared from the alveolus into the interstitium,275–277 and lymphatic drainage subsequently removes the fluid from the interstitium.278,279 Clearance is inhibited by amiloride and ouabain, pharmacologic inhibitors ofsodium (Na+) channels and Na+ pumps, respectively,280,281 emphasizing the importance of Na+ movement in alveolar fluid clearance. Of interest, there are major species differences in both basal and stimulated rates of AFC, although mechanisms responsible for these differences are not entirely clear.275,277,281–290

Publisher Summary

This chapter deals with ion permeation and ion selectivity. Ion channels are membrane proteins found in all domains of cellular life. They are needed in every type of cell and probably in all intracellular membranes as well as the plasma membrane. They have transport functions, facilitate net movements of ions and salts and signaling functions, generate electrical signals, and regulate the cellular free calcium concentration. In each of these roles, ion channels open and close in response to local stimuli and pass a select subset of ions at high rates. Like enzymes, they are regulated catalysts with high substrate specificity and rapid throughput operating at nearly diffusion-limited rates. Ion-selective permeability is necessary for ion channels to play their physiological roles. For electrical signaling it is essential to have two classes of ion channels with different electromotive forces, so that alternating opening of one or the other causes the membrane potential to change between two levels. In many ion channels, the selectivity is not perfect. Ion selectivity is explained using several examples of ligand-gated synaptic ion channels. Furthermore, the principles of mechanisms of ion selectivity are detailed. Finally, the study discusses blocking of ion channels and summarizes some permeant ions and pore blockers for common ion channels. The properties of the same are understood in terms of pore geometry, which is explained.

Diastrophic Dysplasia

Diastrophic dysplasia (MIM 22600) is a well-characterized disorder recognized at birth by the presence of very short extremities, clubfoot, and short hands, with proximal displacement of the thumb producing a hitchhiker appearance (Fig. 717.1). The hands are usually deviated in an ulnar direction. Bony fusion of the metacarpophalangeal joints (symphalangism) is common, as is restricted movement of many joints, including the hips, knees, and elbows. The external ears often become inflamed soon after birth. The inflammation resolves spontaneously, but leaves the ears fibrotic and contracted (cauliflower ear deformity). Many newborns have a cleft palate.

Radiographs reveal short and broad tubular bones with flared metaphyses and flat, irregular epiphyses (Fig. 717.2). The capital femoral epiphyses are hypoplastic, and the femoral heads are broad. The ulnas and fibulas are disproportionately short. Carpal centers may be developmentally advanced; the first metacarpal is typically ovoid, and the metatarsals are twisted medially. There may be vertebral abnormalities, including clefts of cervical vertebral lamina and narrowing of the interpedicular distances in the lumbar spine.

Complications are primarily orthopedic and tend to be severe and progressive. The clubfoot deformity in the newborn resists usual treatments, and multiple corrective surgeries are common. Scoliosis typically develops during early childhood. It often requires multiple surgical procedures to control, and it sometimes compromises respiratory function in older children. Despite the orthopedic problems, patients typically have a normal life span and reach adult heights in the 105-130 cm range, depending on the severity of scoliosis. Growth curves are available for diastrophic dysplasia.

Some patients are mildly affected and exhibit slight short stature and joint contractures, no clubfoot or cleft palate, and correspondingly mild radiographic changes. The mild phenotype tends to recur within families. The recurrence risk of this autosomal recessive condition is 25%. Ultrasonographic examination can be employed for prenatal diagnosis, but ifDTDST mutations can be identified in the patients or parents, molecular genetic diagnosis is possible.

Biological functions

ITP facilitates chloride ion transport from the lumen to the hemolymph and inhibits acid secretion into the lumen in the ileum, leading to the generation of the electrochemical gradient driving water resorption.1 As ITPL inhibits the stimulatory effect on ion transport exerted by ITP, it is proposed that ITPL released from peripheral neurosecretory cells may act as a competitive inhibitor of ITP.3, 7 In Drosophila, ITP (ITP-RE) expression is elevated under osmotic and desiccation stresses, and genetic manipulations of ITP lead to changes in body water content, survival in the above stress conditions, palatability to water and food, and water excretion. These suggest the importance of this hormone as a central neuroendocrine factor integrating water homeostasis.12 Moreover, ITP and ITPL are thought to be involved in various biological processes. In T. castaneum, the knockdown of ITP/ITPL-1/ITPL-2 using double-stranded RNAs in the larval stages causes an increase of mortality in subsequent developmental stages, finally attaining almost 100% around the time of eclosion. Knockdown during the pupal stage results in a significant reduction in the number of eggs produced and in the survival rate of offspring, accompanied by developmental defects of the ovary.4 ITP and/or ITPL possibly contribute to the control of ecdysis.8 Additionally, ITP and ITPL are each supposed to function as a neurotransmitter/neuromodulator as well as a hormone circulating in the hemolymph. For instance, it is suggested that ITP is one of the neurotransmitters released from the clock neurons of the Drosophila brain.13

Diuretic Agents and Ion Transporters

Both weak and high-ceiling diuretics reduce CSF production. The therapeutic strategy is to suppress fluid formation without altering CSF composition, by interfering with choroidal ion transporters at apical or basolateral membranes. One desirable clinical outcome is to lower ICP by decreasing fluid input to the ventricles. To interpret hydrophilic drug effects, a significant factor is whether the inhibiting agent is administered on the blood side (intravenously or intraperitoneally) or CSF side (intraventricularly or intrathecally). Tight junctions between choroid epithelial cells, including tricellular tight junctions,79 limit penetration of water-soluble agents by diffusion across the blood-CSF barrier. Hydrophilic agents such as ouabain and furosemide, potent inhibitors, respectively, of apical Na+-K+-ATPase and Na+-K+-Cl− cotransport, therefore do not reduce CSF formation when presented on the blood side of the choroid plexus barrier.20 Therapeutic drug access to the transporter target is critical.

Dual apical transporter targets are the Na+ pump and the Na+-K+-Cl− cotransporter. Direct inhibition of Na+ pumping is accomplished with cardiac glycosides. Intraventricular ouabain reduces CSF formation80 but elevates CSF K+ concentration20 and is not therapeutically feasible. Digoxin, which is more lipid soluble, permeates the blood-CSF barrier to reach target sites on the ventricular side. Patients treated with digoxin have about a 25% decline in CSF formation.81 Geriatric patients receiving digoxin may have a neurotoxicity risk due to reduced CSF turnover added to an already low-baseline CSF production in senescence.38 Another CSF-facing membrane target is Na+-K+-Cl− cotransport, which is bumetanide sensitive.82 Bumetanide acts on the kidney to reduce peripheral tissue swelling and fluid retention. It has also been tested on choroid plexus, the “kidney” of the CNS.39 When presented intraventricularly (0.1 mM) in dogs, bumetanide curtails CSF production up to 50%.83 Bumetanide given intravenously negligibly affects CSF formation,84 presumably because of poor systemic access to the choroidal apical membrane. Furosemide, another high-ceiling diuretic, also reduces CSF formation and ICP when given through CSF.85 At high doses furosemide alters choroidal blood flow and carbonic anhydrase activity as well as Na+-K+-Cl− cotransport. A third pharmacologic target on the CSF-facing membrane is the Na+-HCO3− cotransporter.54 Awaiting elucidation are the role of this HCO3− cotransporter in CSF formation and the use of novel agents to access it after systemic administration.

Ion Transport Peptide (ITP)

Schgr-ITP (Fig. 1) is the only identified ADH that stimulates fluid reabsorption from the insect hindgut and was isolated from the locust, Schistocerca gregaria, on the basis of its ability to increase electrogenic Cl− transport across the ileum.2 A cDNA encoding the prepropeptide was cloned and the C-terminal amidated peptide has 39–42% sequence similarity to crustacean hyperglycemic hormone (CHH) from the shore crab, Carcinus maenas.24 A splice variant of Schgr-ITP encodes an Schgr-ITP-like peptide (ITP-L) with a nonamidated C-terminus (Fig. 1), which has no currently known biological function. cDNAs encoding ITP and ITP-L have been cloned from M. sexta, B. mori, and A. aegypti.8 Similarly, in Drosophila melanogaster and Tribolium castaneum, ITP and two distinct ITP-L peptides have been identified.32 Only the T. castaneum ITP is not amidated at the C-terminus.

Ion transport is integral to the chemiosmotic theory. In this chapter, we describe the basic permeability properties of membranes and the abilities of ionophores to induce additional pathways of ion permeation, bearing in mind that what is discussed is equally applicable to energy-conserving and non-energy-conserving membranes—after all, the only significant distinction between the two is the presence of the proton pumps in the former. In Part 3, this generality will become apparent when ionophores are seen to insert into all the membranes of intact cells with frequently complex results.

5 Ion Transport in Polymer Electrolytes

Ion transport in polymer electrolytes takes place only in the amorphous, elastomeric phase of the ion–polymer complexes. In PEO–Li+ electrolytes, the motion of Li+ ions is enabled by the simultaneous making and breaking of bonds with the oxygen atoms of the PEO chain, and is assisted by segmental motion of the PEO chain. Therefore, the ion transport in polymer electrolytes is thermally activated and is related to the glass transition phenomena. The temperature dependence of the ionic conductivity can be expressed by the Vogel–Tamman–Fulcher (VTF) equation:

(1)σ=AT−1/2exp−Ea/T−T0

where A is a constant, T is temperature, T0 is the “ideal” glass transition temperature (about 50 °C below the actual glass transition temperature, Tg), and Ea is the activation energy. The strong temperature dependence of ionic conductivity is a general feature. At temperatures below Tg, the ion mobility is negligible because the chain segmental motion is quenched.

This “freeze-out” of the ion mobility allows the stabilization of the dynamic p–i–n junction in LECs by lowering the operating temperature. At temperatures below Tg of the ion transport polymer (about 208 K for PEO), the ions will be immobilized owing to the quenching of the polymer chain segmental motion. Both the doping profile and the p–i–n junction will be stabilized, giving “frozen-junction” LECs.

3.1.2.4 Ion Transport Peptides and Crustacean Hyperglycemic Hormones

Insect ITPs belong to a large family of peptides that are prominent in crustacean physiological functions: crustacean hyperglycemic hormones (CHHs), molt inhibiting hormones (MIHs), vitellogenesis inhibiting hormones (VIHs), and mandibular organ inhibiting hormones (MOIHs) (reviews: de Kleijn et al., 1998; Van Herp, 1998; Webster, 1998; see also Chapter 3.16). As their names indicate, these large peptides regulate various functions including water homeostasis, energy mobilization, vitellogenesis, and biosynthesis of ecdysteroids and methyl farnesoate (Keller, 1992; Audsley et al., 1992a, 1992b; Meredith et al., 1996; Wainwright et al., 1996; Liu et al., 1997; Keller et al., 1999).

Since some actions of CHH are associated with crustacean ecdysis, it is given emphasis here. CHH was originally identified in sinus gland extracts of Carcinus maenas as an amidated 72 amino acid peptide (Kegel et al., 1989). Subsequently, numerous related peptides and corresponding precursors and genes were identified in several crustaceans (Weidemann et al., 1989; Tensen et al., 1991; de Kleijn et al., 1994, 1995; Ohira et al., 1997; Gu and Chan, 1998; Gu et al., 2000).

Peptides of the CHH family control multiple functions. For example, CHH stimulates glucose and lipid release into the hemolymph, secretion of digestive enzymes from the hepatopancreas, and ion transport in the gill. This peptide also inhibits production of ecdysteroids and methyl farnesoate from endocrine glands (Keller, 1992; Van Herp, 1998; Spanings-Pierrot et al., 2000). Immunohistochemistry revealed that, in several crustaceans, CHH is produced by the eyestalk neuroendocrine system composed of neurosecretory cells in the X-organ and neurohemal sinus gland (Dircksen et al., 1988).

Recent studies showed that CHH immunoreactivity also is produced by a novel endocrine system comprising thousands of endocrine cells in the fore- and hindgut of Carcinus (Chung et al., 1999; Webster et al., 2000). Biochemical and molecular evidence confirmed that this gut immunoreactive peptide and its precursor sequence were identical to CHH produced by the eyestalk neurosecretory system (Chung et al., 1999). Massive release of CHH from gut endocrine cells was detected during ecdysis of Carcinus, causing water and ion uptake necessary for swelling of the animal and rupture of the epidermal lines, followed by ecdysis of the old cuticle and the subsequent increase in size during postecdysis (Chung et al., 1999). CHH-related peptides likely control water and ion balance in other arthropods as well, as described in the locust (Phillips et al., 1996, 1998).

Ion transport peptide was originally identified in the storage lobe of locust CC and shown to stimulate salt and water reabsorption, and to inhibit acid secretion by the hindgut of Schistocerca gregaria (Audsley et al., 1992a, 1992b). Western blots with antisera to Schistocerca ITP suggest that this peptide is quickly transported from the brain neurosecretory cells into the neurohemal CC–CA. On Western blots, a strongly stained band corresponding to ITP was detected in extracts of two CC–CA, while only a weakly labeled band was visible in extracts of 50 brains (Macins et al., 1999). Using Schistocerca ileal bioassays and Western blots, evidence for the presence of ITP was detected in the CC or brain extracts of orthopteroid insects, including several locusts, cricket A. domestica, cockroach Periplaneta americana, and stick insect Carausius morosus. By the same criteria, ITP was not detected in holometabolous insects, such as the moth Spodoptera litura and the fly Neobellieria bullata (Macins et al., 1999). Nevertheless, precursors encoding peptides related to ITP/CHH have been identified in both hemi- and holometabolous insects such as Schistocerca, Locusta, and Bombyx (Meredith et al., 1996; Macins et al., 1999; Endo et al., 2000), and gene sequences encoding putative ITPs became generally accessible after publication of the Drosophila and Anopheles genomes. These precursors and genes encode an amidated 72 amino acid ITP and 1–2 longer free C-terminal ITP-like peptides (Figure 8).

ITP immunoreactivity and mRNA encoding the peptide occur in the CNS of moths. In situ hybridization with anti-sense riboprobes detected ITP mRNA in 5–6 pairs of large neurons in the Bombyx brain (Endo et al., 2000). Immunohistochemical staining with CHH and ITP antisera (Figure 9c; Spalovská-Valachová and Zitnan, unpublished data) showed that these neurons are ipsilateral neurosecretory cells (type Ia2). Smaller paired neurons were also stained in each ventral ganglion of Schistocerca and Manduca (Dircksen and Heyn, 1998; Spalovska-Valachova and Zitnan, unpublished data).

At the present time, definitive roles for ITP/CHH-like peptides in insect ecdysis are not well understood. But based on analogy with gut CHH in crustaceans and the demonstrated role of ITP in gut ion transport in locusts, it is speculated that blood-borne ITP may control functions associated with water reabsorption or air inflation, which are necessary preparatory steps for shedding of the old cuticle.

Pumps

Ion pumps are discussed in Chapter 3. Other reviews of ion pumps are Apell (4, 5), Facciotti et al. (23), Fambrough and Inesi (24), Finbow and Harrison (26), Horisberger (48, 49), Läuger (63), and Sachs et al. (87).

Ion pumps can be classified according to the source of metabolic energy. Pumps in general can be driven by light, redox state, or ATP hydrolysis. Animal-cell plasma membrane ATPases belong to the P type, which is characterized by the formation of a phosphorylated intermediary (Na+,K+-, H+,K+-, and Ca2+-ATPase), or to the V type (vacuolar H+-ATPase). In intracellular membranes, there is expression of the vacuolar-type H+ pump, as well as F-type (or F1- or F0-type) ATPases, that is, the ATP synthase expressed in the inner mitochondrial membrane. ATP synthases are also expressed in purple bacteria and in green plants. Their function can be outlined as follows. Multimeric protein complexes (respiratory-chain complex in mitochondria, bacteriorhodopsin in purple bacteria, photosynthetic reaction center in chloroplasts) generate a H+ electrochemical gradient from the redox potential of NADPH (mitochondria) or light energy (others). The transmembrane H+ electrochemical gradient is then used by ATP synthases to synthesize ATP from ADP and PI. Hence, the function of these proteins is to synthesize ATP, dissipating the ion gradient in the process. However, they are reversible, that is, under appropriate conditions they will hydrolyze ATP and generate an electrochemical ion gradient. Similarly, the Na+,K+-ATPase normally hydrolyzes ATP to transport actively Na+ and K+, but under certain conditions can operate in a reverse mode, that is, downhill ion fluxes coupled to the synthesis of ATP.

The ion pumps present in plasma membranes of epithelial cells are the Na+,K+-ATPase, Ca2+-ATPase, H+-ATPase, and H+,K+-ATPase. Other pumps have been suggested to exist in these tissues, but they are either unique to certain epithelia or controversial. The molecular structures of these pumps have been identified and significant progress has been made in understanding their function, but much work remains to be done in this area. Ion occlusion is a well-known stage during the pump cycle (34). As is the case with carrier function, occlusion denotes that the conformational change necessary for transport does change the accessibility of the substrate-binding sites to the solutions bathing the membrane.

The Na+,K+-, H+,K+-, and Ca2+-ATPase are all of the P type, which is characterized by the phosphorylation of an aspartic acid residue (in the sequence DKTG) during the pump catalytic cycle. The Na+,K+-ATPase is a ubiquitous pump in epithelial cells. In each cycle, one ATP molecule is hydrolyzed, three Na+ are transported from the cytoplasm to the interstitial fluid, and two K+ are transported in the opposite direction. In each cycle there is thus a net transfer of charge across the membrane (one net charge is extruded), and hence the pump is electrogenic, tending to hyperpolarize the cell. Its turnover number is in the range of values for carriers, that is, less than 102 s−1. The catalytic cycle of P-type ATPases is shown in Fig. 10.

The Na+,K+-ATPase is expressed in most, if not all, vertebrate epithelial cells. It is generally targeted to the basolateral membrane. In the choroid plexus and the retinal pigment epithelium, the pump is located at the apical membrane and cell polarity appears inverted vis-à-vis other epithelia. The pump consists of α and β subunits in a 1:1 stoichiometry (probably α2β2). The α subunit has four isoforms (apparent molecular mass of 120 kDa) and the β subunit has two isoforms (1 and 2) with apparent molecular mass of 50 kDa. The α subunit is responsible for both ion transport and ATP activity, and contains the Na+,K+ (and ouabain)-binding sites, as well as the phosphorylation site. The β subunit (three isoforms) appears to be required for assembly and plasma membrane targeting of the protein. The pump isoforms are tissue-specific and change during organ development.