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Systematic family-wide analysis of sodium bicarbonate cotransporter NBCn1/SLC4A7 interactions with PDZ scaffold proteins.

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 ?1, ?2, ?2, 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 ?2 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 ?2 and conversely GST/syntrophin ?2 pulled down NBCn1. Moreover, syntrophin ?2 increased intracellular pH recovery, from acidification, mediated by NBCn1's Na/HCO3 cotransport. Syntrophin ?2 also increased an ionic conductance produced by NBCn1 channel-like activity. Thus, syntrophin ?2 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.

SUBMITTER: Lee HJ

PROVIDER: S-EPMC4098744 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Systematic family-wide analysis of sodium bicarbonate cotransporter NBCn1/SLC4A7 interactions with PDZ scaffold proteins.

Lee Hye Jeong HJ Kwon Min Hyung MH Lee Soojung S Hall Randy A RA Yun C Chris CC Choi Inyeong I

Physiological reports 20140520 5

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 PD ...[more]

PMID: 24844638

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Project description:RATIONALE:Salt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure. OBJECTIVE:To characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5. METHODS AND RESULTS:The expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte nuclear factor type 4A (HNF4A) to DNA was increased in hRPTCs carrying HV SLC4A5 rs7571842 but not rs10177833. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was abolished by HNF4A antagonists. CONCLUSION:NBCe2 activity is stimulated by an increase in intracellular sodium and is hyper-responsive in hRPTCs carrying HV SLC4A5 rs7571842 through an aberrant HNF4A-mediated mechanism.

Dataset Information

Systematic family-wide analysis of sodium bicarbonate cotransporter NBCn1/SLC4A7 interactions with PDZ scaffold proteins.

Publications

Systematic family-wide analysis of sodium bicarbonate cotransporter NBCn1/SLC4A7 interactions with PDZ scaffold proteins.

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