Effects of Nt-truncation and coexpression of isolated Nt domains on the membrane trafficking of electroneutral Na+/HCO3– cotransporters

Abstract

The SLC4 genes are all capable of producing multiple variants by alternative splicing or using alternative promoters. The physiological consequences of such diversity are of great interest to investigators. Here, we identified two novel variants of the electroneutral Na+/ cotransporter NBCn1, one full-length starting with “MIPL” and the other Nt-truncated starting with “MDEL”. Moreover, we identified a new promoter of Slc4a10 encoding NBCn2 and a novel type of Nt-truncated NBCn2 starting with “MHAN”. When heterologously expressed, the new NBCn1 variants were well localized to the plasma membrane and exhibited characteristic NBCn1 activity. However, MHAN-NBCn2 was poorly localized on the plasma membrane. By deletion mutations, we identified the Nt regions important for the surface localization of NBCn2. Interestingly, coexpressing the full-length NBCn2 greatly enhances the surface abundance of the Nt-truncated NBCn2. Co-immunoprecipitation and bimolecular fluorescence complementation studies showed that the full-length and Nt-truncated NBCn2 interact with each other to form heterodimers in neuro-2A cells. Finally, we showed that the isolated Nt domain interacts with and enhances the surface abundance of the Nt-truncated NBCn2. The present study expands our knowledge of the NBCn1 and NBCn2 transcriptome, and provides insights into how the Nt domain could affect transporter function by regulating its membrane trafficking.

Na+/HCO3 – Cotransporter NBCn2 Mediates HCO3 − Reclamation in the Apical Membrane of Renal Proximal Tubules

“…In rats, the Slc4a10 gene can give rise to multiple NBCn2 variants that fall into two major groups. Depending on the usage of different promoters, 15 the extreme Nt of these variants can begin either with MCDL (NBCn2-E/F/G/H) or MEIK (NBCn2-A/B/C/D). In the MCDL group, the rat kidney mainly expresses NBCn2-G, and in the MEIK group, it expresses mainly NBCn2-C. 14,15 Here, we find that MCDL-NBCn2 and MEIK-NBCn2 exhibit distinct tissue and subcellular distributions in rat kidney, with MCDL-NBCn2 being localized in the apical membrane of PTs, and MEIK-NBCn2 localized in the BLM of mTALs and IMCDs.…”

“…Previous studies have shown that the rat kidney expresses two groups of full-length NBCn2 (Slc4a10) variants, differing in the amino termini (Nts), one starting with residue MCDL (mainly NBCn2-G), and the other with MEIK (mainly NBCn2-C). 14,15 Here, we report that MCDL-NBCn2 is expressed at the apical membrane of the PT, whereas MEIK-NBCn2 is expressed at the BLM of the medullary thick ascending limb (mTAL) and inner medullary collecting ducts (IMCDs). Systemic acid-base disturbances reciprocally regulate the abundances of MCDL-NBCn2 and NHE3.…”

Structure and Function of SLC4 Family HCO3- Transporters

“…A series of studies have shown that the variable region Nt-VR1 is not necessary for the surface expression and transport activity of NCBTs (McAlear et al, 2006 ; Parker et al, 2007a ; Fukuda et al, 2013 ; Wang et al, 2015 ). When heterologously expressed in Xenopus oocytes, NBCn1-Q, a natural variant of Slc4a7 lacking Nt-VR1, contains minimal transport activity and typical Na + -conductance characteristic of NBCn1 (Wang et al, 2015 ). However, removal of the entire Nt domain almost completely inactivates rat NBCe1-C when heterologously expressed in Xenopus oocytes, although this molecular maneuver has little effect on the surface expression of NBCe1 (McAlear et al, 2006 ).…”

“…The alternative Nts arise from two different types of sources: (1) transcription using distinct promoters, e.g., the generation of alternative Nts of NBCe1-A vs. NBCe1-B (Abuladze et al, 2000 ); (2) alternative splicing of cassette exons in the 5′ portion of the messenger RNAs, e.g., the generation of alternative Nts of NBCn1-Q vs. NBCn1-R (Wang et al, 2015 ). Generally, the alternative Nts have little similarity among different SLC4 family members and different variants of the same member.…”

“…The three NBCe2 variants shown in Figure 5 contain two OSEs: (1) Nt-VR1 that is present in NBCe2-a/c and truncated in NBCe2-g; (2) cassette ε of 16 aa in TMD that is present in NBCe2-a and absent in NBCe2-c and -g. Cassete ε is derived from alternatives splicing of cassette exon 27 (see review by Parker and Boron, 2013 ). Reminiscent of kAE1, NBCe2-g lacking the entire Nt-VR1 is well expressed on the plama membrane and functionally active (Fukuda et al, 2013 ; Wang et al, 2015 ), demonstrating that Nt-VR1 is dispensable for the transport activity of NBCe2. The functional significance of cassette ε remains unclear.…”