Volume 16, Issue 3, Pages 331-341 (November 2004) NaBC1 Is a Ubiquitous Electrogenic Na+-Coupled Borate Transporter Essential for Cellular Boron Homeostasis and Cell Growth and Proliferation  Meeyoung Park, Qin Li, Nikolay Shcheynikov, Weizong Zeng, Shmuel Muallem  Molecular Cell  Volume 16, Issue 3, Pages 331-341 (November 2004) DOI: 10.1016/j.molcel.2004.09.030

Figure 1 Electrogenic H+/OH− and Na+ Transport by NaBC1 in the Absence of Borate (A–E) HEK293 cells were transfected with GFP (controls [A, C, and F]) or GFP and NaBC1 (B, D, E, and G–I). The cells in (A)–(E) were loaded with BCECF to measure pHi. Where indicated by the bars, the cells were incubated in Na+-free media (A–E) and 0.1 or 10 μM EIPA (A and B). All solutions used for the experiments in (C)–(I) contained 0.1 μM EIPA to inhibit the endogenous NHE1 activity. In (C) and (D), the cells were exposed to bath solutions of pHo 7.4, 6.5, or 8.2. In (E), cells incubated in media containing 5, 0, or 100 mM K+ were incubated in Na+-free media. The shaded area marks the pHi changes evoked by changing the membrane potential in the continuous absence of Na+o. The models at bottom left indicate the two transport modes that can explain H+/Na+/OH− transport by NaBC1. (F–I) The cells in (F)–(I) were loaded with SBFI to measure Na+i. In (F) and (G), the cells were exposed to solutions containing 145 mM Na+ and buffered to pHo of 6.5 or 8.5 and then to Na+-free media, as indicated by the bars. In (H) and (I), the cells were depolarized to various degrees by incubation in solutions containing 100, 60, or 40 mM K+, as indicated, and then in Na+-free media. Note that membrane depolarization resulted in Na+ efflux only in cells expressing NaBC1. Molecular Cell 2004 16, 331-341DOI: (10.1016/j.molcel.2004.09.030)

Figure 2 Borate Transport by NaBC1 The model depicts the mechanism of borate homeostasis in animal cells and the possible role of NaBC1 in this process. HEK293 cells transfected with GFP (controls, A) or GFP and NaBC1 (B–D) were used to measure pHi. The cells were incubated with the indicated concentrations of borate between 0–10 mM before perfusion in Na+-free media (A and B). In (C), the cells were incubated in solutions containing between 5 and 20 mM arsenate or 5 mM borate before perfusion with Na+-free media. Note that NaBC1 does not transport the closely similar metalloid arsenate. In (D), the cells were incubated with solutions whose pHo was buffered to 7.4, 8.2, or 7.0 in the absence (first portion of experiment) or presence of 5 mM borate. Note that borate markedly attenuated the increase in pHi and increased the acidification in response to pHo of 8.2 and 7.0, respectively. pHo of 7.0 was used since at lower pHo and in the presence of borate the dye was saturated. Molecular Cell 2004 16, 331-341DOI: (10.1016/j.molcel.2004.09.030)

Figure 3 Na+-B(OH)4− Current by NaBC1 The whole-cell current was measured in nontransfected cells (A) and HEK293 cells transfected with NaBC1 (B and C) incubated in solution A containing 145 mM Na+ (dashed traces) that also contained 25 mM borate (gray traces), or Na+-free media containing 25 mM borate (black traces). In (C), the borate-dependent current was isolated by subtracting the current recorded in the absence of borate. Note the pA scale for the inward (gray) and the nA scale for the outward (black) currents. Control, water injected, oocytes (D, G, and H) and oocytes injected with NaBC1 cRNA (E, I, and J) were used to measure the current in media containing Na+ (□), Na+ and 25 mM borate (•), or Na+-free and 25 mM borate (▴). (D) and (E) show the I/V curves. (F) shows the voltage protocol used to obtain the instantaneous current traces in (G)–(J). Note the strong outward rectification of the current in (C) and (E) and the time-dependent activation of the current at high depolarizing voltages in (H) and (J), indicating regulation of NaBC1 by membrane voltage. Molecular Cell 2004 16, 331-341DOI: (10.1016/j.molcel.2004.09.030)

Figure 4 Na+o and Na+i Dependence of OH− and B(OH)4− Transport and Na+ Current by NaBC1 (A) pHi was measured in HEK293 cells transfected with NaBC1 and perfused with media containing 0.1 μM EIPA, the Na+o concentrations indicated next to the bars, and in the absence and then in the presence of 5 mM borate (gray bar). (B) Summary of results obtained in four experiments similar to that in (A) by plotting the extent of acidification as a function of Na+o. Note the reverse Na+o scale and the large shift in Na+o dependence in the presence of borate. (C) The cells were incubated in Na+-free media for 25 min to deplete Na+i before addition of borate (second gray bar). In additional experiments, we found that the extent of acidification by borate was a function of the incubation time in Na+-free media (data not shown). (D and E) Cells transfected with NaBC1 and incubated in Na+-free media were perfused with a Na+-free pipette solution (gray traces) or a pipette solution containing 30 mM Na+ (black traces), and the whole-cell current was measured in the absence (D) or presence of 25 mM borate (E). Note the different current scales in (D) and (E). Molecular Cell 2004 16, 331-341DOI: (10.1016/j.molcel.2004.09.030)

Figure 5 Immunoblotting, Immunolocalization, and Activity of Native NaBC1 (A) Western blot analysis of NaBC1 in extracts prepared from untransfected HEK293 cells (left blot) and HEK293 cells transfected with NaBC1 and the indicated, untransfected cell lines (right blot). The blots were probed with anti-NaBC1 or anti-NaBC1 antibodies preadsorbed with the peptide used to raise the antibodies (αC1+pep). (B) Extracts prepared from the indicated tissues were used to immunoprecipitate NaBC1, and the imunoprecipitated proteins were probed for NaBC1. The lane labeled NaBC1 is a control of an extract from HEK293 cells expressing NaBC1. (C–E) Immunolocalization of NaBC1 in SMG (C and D) and kidney (E) sections. In the control of (C), the anti-NaBC1 antibodies were preincubated with the peptide used to raise the antibodies prior to use. Note that NaBC1 is expressed in the basolateral membrane of SMG acini and most kidney tubules and that acini express a higher level of NaBC1 than the ducts. (F and G) Borate transport in SMG acini (F) and ducts (G) was evaluated from measurements of pHi. Acini express only NHE1, and thus most of their Na+/H+ exchange activity was inhibited by 0.2 μM EIPA. Borate between 2.5 and 10 mM induces a prominent Na+-B(OH)4− transport, as is evident from the extent of acidification in Na+-free media (F). The ducts express NHE1, 2, and 3 and have a much more prominent Na+/H+ exchange activity that was largely inhibited by 10 μM EIPA. The same protocols used with acini show a lower Na+-B(OH)4− transport in the ducts (G) compared to acini, consistent with their low expression of NaBC1 protein (D). Molecular Cell 2004 16, 331-341DOI: (10.1016/j.molcel.2004.09.030)

Figure 6 Borate Stimulates the MAPK Pathway and Cell Growth and Proliferation (A and B) Cell growth was analyzed by cell counting as detailed in the Experimental Procedures. After serum starvation and synchronization, HEK293 (A) or HeLa (B) cells in 24-well plates were treated with the indicated concentrations of borate, and cells were counted 16 hr after treatment. (C) A similar protocol was used to treat control (□) or NaBC1-transfected HeLa cells (•) for measurement of thymidine incorporation, as detailed in the Experimental Procedures. Expression of NaBC1 increased the apparent affinity for the effect of borate on cell growth. (D) Activation of the MAPK pathway was tested by incubating HeLa cells with 0, 0.3, or 5 mM borate for 1, 5, or 60 min. The extracts were analyzed for expression of total MEK1/2 and ERK1/2 and for phosphorylated pMEK1/2 and pERK1/2, as indicated. Similar results were obtained in three experiments. (E) Inhibition of MEK1/2 with 2 (middle columns) or 10 μM (right columns) U0126 inhibited borate-stimulated thymidine incorporation. *p < 0.05. Molecular Cell 2004 16, 331-341DOI: (10.1016/j.molcel.2004.09.030)

Figure 7 NaBC1 Is Essential for Cell Growth and Proliferation (A and B) HeLa cells were treated with luciferase siRNA (control) and the three NaBC1-siRNAs listed in the Experimental Procedures for 72 hr. RNA and proteins were extracted from the same cells and analyzed by RT-PCR for the level of NaBC1 mRNA (A) and by blotting for NaBC1 protein (B). In all similar experiments, siRNA1 had a small effect, siRNA2 had an intermediate effect, and siRNA3 was consistently the most effective in reducing NaBC1 protein expression. (C) Subconfluent HeLa cells were treated with the indicated siRNA for 48 or 72 hr and during the last 3 hr were incubated with [3H]thymidine to measure the effect of siRNA on cell proliferation. The results are mean ± SEM of three experiments. *p < 0.05. (D) Cells were treated with siRNA3 for 24 hr, and then control and siRNA-treated cells were incubated with 0.5 mM borate for an additional 48 hr before measurements of [3H]thymidine uptake as above. Molecular Cell 2004 16, 331-341DOI: (10.1016/j.molcel.2004.09.030)