Human SLC4A11-C functions as a DIDS-stimulatable H+(OH−) permeation pathway: partial correction of R109H mutant transport

The SLC4A11 gene mutations cause a variety of genetic corneal diseases, including congenital hereditary endothelial dystrophy 2 (CHED2), Harboyan syndrome, some cases of Fuchs' endothelial dystrophy (FECD), and possibly familial keratoconus. Three NH2-terminal variants of the human SLC4A11 gene, named SLC4A11-A, -B, and -C are known. The SLC4A11-B variant has been the focus of previous studies. Both the expression of the SLC4A11-C variant in the cornea and its functional properties have not been characterized, and therefore its potential pathophysiological role in corneal diseases remains to be explored. In the present study, we demonstrate that SLC4A11-C is the predominant SLC4A11 variant expressed in human corneal endothelial mRNA and that the transporter functions as an electrogenic H+(OH−) permeation pathway. Disulfonic stilbenes, including 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS), 4,4′-diisothiocyanatodihydrostilbene-2,2′-disulfonate (H2DIDS), and 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonate (SITS), which are known to bind covalently, increased SLC4A11-C-mediated H+(OH−) flux by 150–200% without having a significant effect in mock-transfected cells. Noncovalently interacting 4,4′-diaminostilbene-2,2′-disulfonate (DADS) was without effect. We tested the efficacy of DIDS on the functionally impaired R109H mutant (SLC4A11-C numbering) that causes CHED2. DIDS (1 mM) increased H+(OH−) flux through the mutant transporter by ∼40–90%. These studies provide a basis for future testing of more specific chemically modified dilsulfonic stilbenes as potential therapeutic agents to improve the functional impairment of specific SLC4A11 mutant transporters.

Pharmacology of CFTR Chloride Channel Activity

Schultz, B. D., A. K. Singh, D. C. Devor, and R. J. Bridges. Pharmacology of CFTR Chloride Channel Activity. Physiol. Rev. 79, Suppl.: S109–S144, 1999. — The pharmacology of cystic fibrosis transmembrane conductance regulator (CFTR) is at an early stage of development. Here we attempt to review the status of those compounds that modulate the Cl−channel activity of CFTR. Three classes of compounds, the sulfonylureas, the disulfonic stilbenes, and the arylaminobenzoates, have been shown to directly interact with CFTR to cause channel blockade. Kinetic analysis has revealed the sulfonylureas and arylaminobenzoates interact with the open state of CFTR to cause blockade. Suggestive evidence indicates the disulfonic stilbenes act by a similar mechanism but only from the intracellular side of CFTR. Site-directed mutagenesis studies indicate the involvement of specific amino acid residues in the proposed transmembrane segment 6 for disulfonic stilbene blockade and segments 6 and 12 for arylaminobenzoate blockade. Unfortunately, these compounds (sulfonylureas, disulfonic stilbenes, arylaminobenzoate) also act at a number of other cellular sites that can indirectly alter the activity of CFTR or the transepithelial secretion of Cl−. The nonspecificity of these compounds has complicated the interpretation of results from cellular-based experiments. Compounds that increase the activity of CFTR include the alkylxanthines, phosphodiesterase inhibitors, phosphatase inhibitors, isoflavones and flavones, benzimidazolones, and psoralens. Channel activation can arise from the stimulation of the cAMP signal transduction cascade, the inhibition of inactivating enzymes (phosphodiesterases, phosphatases), as well as the direct binding to CFTR. However, in contrast to the compounds that block CFTR, a detailed understanding of how the above compounds increase the activity of CFTR has not yet emerged.

Heterologous expression of rat NHE4: a highly amiloride-resistant Na+/H+ exchanger isoform

Molecular cloning and expression have previously defined three members of the Na+/H+ exchanger (NHE) gene family NHE1 and NHE2 are sensitive to inhibition by amiloride and its 5'-amino alkyl-substituted analogues, whereas NHE3 is quite resistant to amiloride inhibition. Each of these exchangers has narrowly defined cation specificities for Na+ and Li+. Expression studies with NHE4 have not been as successful, with only a description of modest expression of activity (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao, and E. B. Chang. J. Biol. Chem. 269: 29704-29709, 1994). We now report that NHE4 activity in stably transfected fibroblasts is activated by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), permitting functional characterization of this NHE isoform. The activating effect of DIDS was unique among the disulfonic stilbenes, and competition studies suggested a cross-linking mechanism. NHE4 is extremely resistant to amiloride and ethylisopropylamiloride inhibition and, unlike other NHE isoforms, affects K+/H+ exchange as well as Na+/H+ and Li+/H+ exchange. These findings demonstrate that NHE4 is a functionally distinct member of the NHE gene family and suggest a unique physiological role for this cation/H+ exchanger.

Sodium-bicarbonate cotransport in guinea pig ileal crypt cells

Prior studies show that ileal HCO3- secretion is of crypt origin, possibly involving Na+-HCO3- cotransport. To test for the latter, we isolated crypt cells from guinea pig ileum and determined effects of medium HCO3-, Na+, K+, disulfonic stilbenes, and gramicidin on intracellular pH [pHi;2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein fluorescence], cell volume (electronic sizing), and Na+ efflux from 22Na+ -preloaded cells. Ileal crypt cells alkalinized when placed in sodium gluconate-HCO3- medium containing N-5-methyl-5-isobutyl amiloride (1 microM), bumetanide (10 microM) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (250 microM which blocks Cl-/HCO3- exchange but not Na+ dependent HCO3- uptake). Depolarization with either gramicidin (50 microM) or 50 mM K+ caused a further 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-inhibitable increase in pHi. Gramicidin also caused SITS-inhibitable cell swelling. Both gramicidin effects were Na+ dependent: at 0 mM Na+, gramicidin acidified and did not alter cell volume; at 25 mM, gramicidin also acidified; at 90 and 140 mM, gramicidin alkalinized and induced cell swelling. HCO3- -dependent SITS-inhibitable Na+ efflux from 22Na+ -preloaded cells was also seen. We conclude that ileal crypt cells engage in electrogenic Na+ -HCO3- symport.

Characteristics of apical Cl-HCO3 exchanger of bicarbonate-secreting cells in turtle bladder

The apical anion exchanger of the beta-carbonic anhydrase (CA) cells differs from the basolateral exchanger of the alpha-cells by reduced sensitivity to disulfonic stilbenes and lack of immunoreactivity with antibodies to erythrocyte band 3 protein. To characterize the exchanger, we examined the effects on electroneutral bicarbonate secretion (JHCO3n) of Cl- replacement by gluconate, Br-, SO4(2-), and NO3- and of inhibition by 1) acetazolamide (ACZ) with and without pretreatment with sodium azide (NaN3), 2) furosemide, and 3) alpha-cyano-4-hydroxycinnamate (CHC). The Cl-dependent JHCO3n was 0.90 +/- 0.09 mumol/h, similar to the ACZ-inhibitable rate of 0.83 +/- 0.08 mumol/h with an apparent Km for Cl near 3.4 mM. Maximal JHCO3n was comparable at luminal pH 6.8 and 4.5. JHCO3n was reduced to approximately 21% in Br-, 13% in SO4(2-), and 7% in NO3- solutions compared with the rates in chloride solutions. ACZ inhibition was not abolished by pretreatment with NaN3. JHCO3n was only slightly inhibited (14%) by furosemide and not inhibited by CHC. In conclusion, the apical exchanger is selective for chloride and relatively resistant to inhibitors. Its dependence on luminal chloride is such that its transport rate is closely regulated by mucosal chloride at concentrations below 20 mM.