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

Abstract

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.

Human SLC4A11 Is a Novel NH3/H+ Co-transporter

“…Analysis of data from more cells (Fig. 6A) shows that the resting pH i was significantly lower in SLC4A11-PS120 Col4 (pH i 6.98 Ϯ 0.08, n ϭ 20) relative to EV-PS120 (pH i 7.32 Ϯ 0.08, n ϭ 23), suggesting that SLC4A11 provides some H ϩ permeability in the absence of NH 3 , where a negative membrane potential could drive H ϩ influx, as proposed in a recent report (12). Figs.…”

“…2A and 5A, however, do not reveal any measureable H ϩ currents in the absence of NH 3 . Nevertheless, increased H ϩ permeability is evident in SLC4A11-transfected cells during the acid recovery following NH 4 Cl pulses in previous reports (9,12). Fig.…”

“…Interestingly, the effect on baseline pH i is made most evident in PS120 cells because of the lack of Na ϩ /H ϩ exchangers. Previous studies show that when a more robust bicarbonate buffering system is present, baseline pH i differences disappear (9,10,12). This suggests that SLC4A11 H ϩ permeability in the absence of ammonia is a relatively small contributor to pH i regulation, whereas ammonia transport through SLC4A11 is of more physiological relevance.…”

“…In addition, it has been reported that DIDS has no effect on SLC4A11 Na ϩ -H ϩ permeability (10), whereas another group reported that DIDS enhances SLC4A11 H ϩ permeability (12). Therefore, we tested whether these two compounds affect the SLC4A11 NH 3 /H ϩ current in our model system.…”

“…These studies suggested ethyl-isopropylamiloride (EIPA) 3 -sensitive Na ϩ and H ϩ (or OH Ϫ ) permeability through SLC4A11; however, a precise stoichiometry could not be determined. A more recent report has suggested H ϩ permeability through SLC4A11 but independent of Na ϩ (12). Interestingly, ammonium permeability (9) and water permeability (13) have also been suggested for SLC4A11, and the across-species highly conserved asparagine-proline-X (NPX) water channel motif has been identified in a transmembrane domain of SLC4A11 (N 639 PS) (13).…”