May 1, 2014;
Systematic family-wide analysis of sodium bicarbonate cotransporter NBCn1/SLC4A7 interactions with PDZ scaffold proteins.
(SLC4A7) plays a role in transepithelial HCO3 (-) movement and intracellular pH maintenance in many tissues. In this study, we searched PDZ proteins capable of binding to NBCn1
. We screened a protein array membrane, on which 96 different class I PDZ protein peptides were blotted, with the C-terminal domain of NBCn1
fused to GST. Thirteen proteins were identified in these screens: MAGI-3, NHERF
-95, chapsyn-110, ERBIN
-1, densin-180, syntrophins α1, β2, γ2, MUPP1, and PDZK1
. After determining these binding partners, we analyzed the database of known and predicted protein interactions to obtain an NBCn1
interaction network. The network shows NBCn1
being physically and functionally associated with a variety of membrane and cytosolic proteins via the binding partners. We then focused on syntrophin γ2 to examine the molecular and functional interaction between NBCn1
and one of the identified binding partners in the Xenopus oocyte
expression system. GST/NBCn1
pulled down syntrophin γ2 and conversely GST/syntrophin γ2 pulled down NBCn1
. Moreover, syntrophin γ2 increased intracellular pH recovery, from acidification, mediated by NBCn1''s Na/HCO3 cotransport. Syntrophin γ2 also increased an ionic conductance produced by NBCn1
channel-like activity. Thus, syntrophin γ2 regulates NBCn1
activity. In conclusion, this study demonstrates that NBCn1
binds to many PDZ proteins, which in turn may allow the transporter to associate with other physiologically important proteins.
[+] show captions
Figure 1. A proteomic analysis to identify PDZ proteins capable of binding to NBCn1. (A) A proteomic array containing 96 class I PDZ domains from 55 different proteins was screened with the C‐terminal domain of NBCn1 fused to GST. The domains bound to GST/NBCn1 were detected by the anti‐GST antibody. (B) The control experiment with GST only. (C) The PDZ domains spotted on the membrane are listed. The proteins binding to NBCn1 are MAGI‐3 (B2 in the array), NHERF‐1 (B3), NHERF‐2 (B6), PSD‐95 (B8), chapsyn‐110 (C8), ERBIN (C10), MALS‐1 (D10), densin‐180 (E1), syntrophins α1 (E8), β2 (E10), γ2 (E12), MUPP1 (F5), and PDZK1 (G2).
Figure 2. NBCn1 interaction network. The interaction network was built using the STRING program that searches known and predicted protein interactions based on physical and functional associations. The search parameters are described in the Materials and Methods. The interactions between NBCn1 and identified binding proteins (thick black lines) are supported from the array experiments, and the interactions involving individual binding proteins (blue lines) are drawn from experiments‐based evidence in the database. In addition, physical and functional associations among proteins are shown (differently colored lines). Proteins were named according to the human gene/protein name.
Figure 3. Interaction between NBCn1 and syntrophin γ2. (A) Lysates of Xenopus oocytes expressing syntrophin γ2 or none were incubated with GST/NBCn1 fusion proteins containing the C‐terminal amino acids of NBCn1. GST only served as a control. Pull‐down samples were immunoblotted with the syntrophin γ2 antibody. Syntrophin γ2 (60 kDa) was detected in pull–down samples from syntrophin γ2‐expressing oocytes, but not from control oocytes. Lysates were prepared from membrane (M) and cytosol (C). (B) Lysates of oocytes expressing rat NBCn1 were incubated with GST only, GST/Syn‐PDZ containing amino acid residues 1‐231 of syntrophin γ2, which include the PDZ domain, and GST/Syn‐ΔPDZ containing residues 232–539, which include the peckstrin homology domain. NBCn1 was pulled down by GST/Syn‐PDZ, but not by GST/Syn‐ΔPDZ. One of three experiments is shown. (C) Membrane lysates of rat brains were incubated with GST only, GST/Syn‐full containing the full‐length syntrophin γ2, GST/Syn‐PDZ, or GST/Syn‐ΔPDZ. NBCn1 was pulled down by GST/Syn‐full and GST/Syn‐PDZ, but not by GST/Syn‐ΔPDZ.
Figure 4. Effect of syntrophin γ2 on NBCn1‐mediated pHi recovery in Xenopus oocytes. (A–C) Representative pHi traces in oocytes expressing syntrophin γ2, NBCn1, and NBCn1/syntrophin γ2. Oocytes were superfused with HCO3−/CO2‐free ND96 solution and then with a solution equilibrated with 25 mmol/L HCO3−, 5% CO2. pHi was measured with a proton‐selective glass electrode. (D) Mean pHi recovery rate. The recovery rate (dpHi/dt) was calculated from a linear regression line during the first 2 min of recovery (n = 5 for each).
Figure 5. Effect of syntrophin γ2 on NBCn1 conductance. (A) Representative current–voltage (I–V) relationships in a syntrophin γ2‐expressing oocyte or an uninjected oocyte. Oocytes were clamped at −60 mV and subjected to the voltage command stepping from −140 to +40 mV. Steady‐state currents were recorded (n =5 for each). Recordings were done in HCO3−/CO2‐free ND96 solution. (B) I–V relationships in oocytes expressing NBCn1 and different amounts of syntrophin γ2. Large inward currents at negative potentials and outward currents at positive potentials are hallmarks for NBCn1 conductance. (C) Mean slope conductance. Slopes were determined near zero‐current voltages in I–V plots. Data were averaged from uninjected control oocytes (n =12), oocytes expressing syntrophin γ2 (n =16), NBCn1 (n =15), NBCn1 plus syntrophin γ2 at 2 ng (n =17), 10 ng (n =15), and 20 ng (n =24). n.s, not significant. P‐values were calculated to compare each NBCn1/syntrophin with NBCn1 only.