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Physiol Rep
2014 May 20;25:. doi: 10.14814/phy2.12016.
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Systematic family-wide analysis of sodium bicarbonate cotransporter NBCn1/SLC4A7 interactions with PDZ scaffold proteins.
Lee HJ
,
Kwon MH
,
Lee S
,
Hall RA
,
Yun CC
,
Choi I
.
Abstract
NBCn1 (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-1, NHERF-2, PSD-95, chapsyn-110, ERBIN, MALS-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.
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 Câterminal domain of NBCn1 fused to GST. The domains bound to GST/NBCn1 were detected by the antiâGST antibody. (B) The control experiment with GST only. (C) The PDZ domains spotted on the membrane are listed. The proteins binding to NBCn1 are MAGIâ3 (B2 in the array), NHERFâ1 (B3), NHERFâ2 (B6), PSDâ95 (B8), chapsynâ110 (C8), ERBIN (C10), MALSâ1 (D10), densinâ180 (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 experimentsâbased 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 Câterminal amino acids of NBCn1. GST only served as a control. Pullâdown samples were immunoblotted with the syntrophin γ2 antibody. Syntrophin γ2 (60 kDa) was detected in pullâdown samples from syntrophin γ2âexpressing 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/SynâPDZ containing amino acid residues 1â231 of syntrophin γ2, which include the PDZ domain, and GST/SynâÎPDZ containing residues 232â539, which include the peckstrin homology domain. NBCn1 was pulled down by GST/SynâPDZ, but not by GST/SynâÎPDZ. One of three experiments is shown. (C) Membrane lysates of rat brains were incubated with GST only, GST/Synâfull containing the fullâlength syntrophin γ2, GST/SynâPDZ, or GST/SynâÎPDZ. NBCn1 was pulled down by GST/Synâfull and GST/SynâPDZ, but not by GST/SynâÎPDZ.
Figure 4. Effect of syntrophin γ2 on NBCn1âmediated pHi recovery in Xenopus oocytes. (AâC) Representative pHi traces in oocytes expressing syntrophin γ2, NBCn1, and NBCn1/syntrophin γ2. Oocytes were superfused with HCO3â/CO2âfree ND96 solution and then with a solution equilibrated with 25 mmol/L HCO3â, 5% CO2. pHi was measured with a protonâselective 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 currentâvoltage (IâV) relationships in a syntrophin γ2âexpressing oocyte or an uninjected oocyte. Oocytes were clamped at â60 mV and subjected to the voltage command stepping from â140 to +40 mV. Steadyâstate currents were recorded (n =5 for each). Recordings were done in HCO3â/CO2âfree ND96 solution. (B) IâV 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 zeroâcurrent voltages in IâV 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. Pâvalues were calculated to compare each NBCn1/syntrophin with NBCn1 only.
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