Journal article
PIP2 hydrolysis stimulates the electrogenic Na+–bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus laevis oocytes
The Journal of physiology, Vol.590(Pt 23), pp.5993-6011
09/10/2012
DOI: 10.1113/jphysiol.2012.242479
PMCID: PMC3530112
PMID: 22966160
Abstract
Electrogenic Na
+
–bicarbonate cotransporter NBCe1 variants contribute to pH
i
regulation, and promote ion reabsorption or secretion by many epithelia. Most Na
+
-coupled bicarbonate transporter (NCBT) families such as NBCe1 contain variants with differences primarily at the cytosolic N and/or C termini that are likely to impart on the transporters different modes of regulation. For example, N-terminal regions of NBCe1 autoregulate activity. Our group previously reported that cytosolic phosphatidylinositol 4,5-bisphosphate (PIP
2
) stimulates heterologously expressed rat NBCe1-A in inside-out macropatches excised from
Xenopus laevis
oocytes. In the current study on whole oocytes, we used the two-electrode voltage-clamp technique, as well as pH- and voltage-sensitive microelectrodes, to characterize the effect of injecting PIP
2
on the activity of heterologously expressed NBCe1-A, -B, or -C. Injecting PIP
2
(10 μ
m
estimated final) into voltage-clamped oocytes stimulated NBC-mediated, HCO
3
−
-induced outward currents by >100% for the B and C variants, but not for the A variant. The majority of this stimulation involved PIP
2
hydrolysis and endoplasmic reticulum (ER) Ca
2+
release. Stimulation by PIP
2
injection was mimicked by injecting IP
3
, but inhibited by either applying the phospholipase C (PLC) inhibitor U73112 or depleting ER Ca
2+
with prolonged thapsigargin/EGTA treatment. Stimulating the activity of store-operated Ca
2+
channels (SOCCs) to trigger a Ca
2+
influx mimicked the PIP
2
/IP
3
stimulation of the B and C variants. Activating the endogenous G
q
protein-coupled receptor in oocytes with lysophosphatidic acid (LPA) also stimulated the B and C variants in a Ca
2+
-dependent manner, although via an increase in surface expression for the B variant. In simultaneous voltage-clamp and pH
i
studies on NBCe1-C-expressing oocytes, LPA increased the NBC-mediated pH
i
-recovery rate from a CO
2
-induced acid load by ∼80%. Finally, the general kinase inhibitor staurosporine completely inhibited the IP
3
-induced stimulation of NBCe1-C. In summary, injecting PIP
2
stimulates the activity of NBCe1-B and -C expressed in oocytes through an increase in IP
3
/Ca
2+
that involves a staurosporine-sensitive kinase. In conjunction with our previous macropatch findings, PIP
2
regulates NBCe1 through a dual pathway involving both a direct stimulatory effect of PIP
2
on at least NBCe1-A, as well as an indirect stimulatory effect of IP
3
/Ca
2+
on the B and C variants.
Details
- Title: Subtitle
- PIP2 hydrolysis stimulates the electrogenic Na+–bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus laevis oocytes
- Creators
- Ian M Thornell - Institut de Biologie Intégrative de la CelluleJianping Wu - Institut de Biologie Intégrative de la CelluleXiaofen Liu - Institut de Biologie Intégrative de la CelluleMark O Bevensee - Institut de Biologie Intégrative de la Cellule
- Resource Type
- Journal article
- Publication Details
- The Journal of physiology, Vol.590(Pt 23), pp.5993-6011
- Publisher
- Blackwell Science Inc
- DOI
- 10.1113/jphysiol.2012.242479
- PMID
- 22966160
- PMCID
- PMC3530112
- ISSN
- 0022-3751
- eISSN
- 1469-7793
- Language
- English
- Date published
- 09/10/2012
- Academic Unit
- Pulmonary, Critical Care, and Occupational Medicine; Internal Medicine
- Record Identifier
- 9984359574902771
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