Journal article
Phosphatidylinositol 4,5-bisphosphate degradation inhibits the Na+/bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus oocytes
The Journal of physiology, Vol.593(3), pp.541-558
02/01/2015
DOI: 10.1113/jphysiol.2014.284307
PMCID: PMC4324704
PMID: 25398525
Abstract
Key points We previously reported that the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) directly stimulates heterologously expressed electrogenic Na+/bicarbonate cotransporter NBCe1-A in an excised macropatch from the Xenopus oocyte, and indirectly stimulates NBCe1-B and -C in the intact oocyte primarily through inositol 1,4,5-trisphosphate/Ca2+. In the current study, we expand on a previous observation that PIP2 may also directly stimulate NBCe1 in the intact oocyte. In this study on oocytes, we co-expressed either NBCe1-B or -C and a voltage-sensitive phosphatase (VSP), which depletes PIP2 without changing inositol 1,4,5-trisphosphate, and monitored NBCe1-mediated currents with the two-electrode voltage-clamp technique or pH(i) changes using V-m/pH-sensitive microelectrodes. Activating VSP inhibited NBCe1-B and -C outward currents and NBCe1-mediated pH(i) increases, and changes in NBCe1 activity paralleled changes in surface PIP2. This study is a quantitative assessment of PIP2 itself as a regulator of NBCe1-B and -C in the intact cell, and represents the first use of VSP to characterize the PIP2 sensitivity of a transporter. These data combined with our previous work demonstrate that NBCe1-B and -C are regulated by two PIP2-mediated signalling pathways. Specifically, a decrease in PIP(2)per se can inhibit NBCe1, whereas hydrolysis of PIP2 to inositol 1,4,5-trisphosphate/Ca2+ can stimulate the transporter.
The electrogenic Na+/bicarbonate cotransporter (NBCe1) of the Slc4 gene family is a powerful regulator of intracellular pH (pH(i)) and extracellular pH (pH(o)), and contributes to solute reabsorption and secretion in many epithelia. Using Xenopus laevis oocytes expressing NBCe1 variants, we have previously reported that the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) directly stimulates NBCe1-A in an excised macropatch, and indirectly stimulates NBCe1-B and -C in the intact oocyte primarily through inositol 1,4,5-trisphosphate (InsP(3))/Ca2+. In the current study, we used the two-electrode voltage-clamp technique alone or in combination with pH/voltage-sensitive microelectrodes or confocal fluorescence imaging of plasma membrane PIP2 to characterize the PIP2 sensitivity of NBCe1-B and -C in whole oocytes by co-expressing a voltage-sensitive phosphatase (VSP) that decreases PIP2 and bypasses the InsP(3)/Ca2+ pathway. An oocyte depolarization that activated VSP only transiently stimulated the NBCe1-B/C current, consistent with an initial rapid depolarization-induced NBCe1 activation, and then a subsequent slower VSP-mediated NBCe1 inhibition. Upon repolarization, the NBCe1 current decreased, and then slowly recovered with an exponential time course that paralleled PIP2 resynthesis as measured with a PIP2-sensitive fluorophore and confocal imaging. A subthreshold depolarization that minimally activated VSP caused a more sustained increase in NBCe1 current, and did not lead to an exponential current recovery following repolarization. Similar results were obtained with oocytes expressing a catalytically dead VSP mutant at all depolarized potentials. Depleting endoplasmic reticulum Ca2+ did not inhibit the NBCe1 current recovery following repolarization from VSP activation, demonstrating that changes in InsP(3)/Ca2+ were not responsible. This study demonstrates for the first time that depleting PIP(2)per se inhibits NBCe1 activity. The data in conjunction with previous findings implicate a dual PIP2 regulatory pathway for NBCe1 involving both PIP2 itself and generated InsP(3)/Ca2+.
Details
- Title: Subtitle
- Phosphatidylinositol 4,5-bisphosphate degradation inhibits the Na+/bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus oocytes
- Creators
- Ian M. Thornell - University of Alabama at BirminghamMark O. Bevensee - University of Alabama at Birmingham
- Resource Type
- Journal article
- Publication Details
- The Journal of physiology, Vol.593(3), pp.541-558
- Publisher
- Wiley
- DOI
- 10.1113/jphysiol.2014.284307
- PMID
- 25398525
- PMCID
- PMC4324704
- ISSN
- 0022-3751
- eISSN
- 1469-7793
- Number of pages
- 18
- Grant note
- R01NS046653 / NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Neurological Disorders & Stroke (NINDS) T32 GM008111 / NIH/NIGMS; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of General Medical Sciences (NIGMS) R01 NS046653 / NIH/NINDS; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Neurological Disorders & Stroke (NINDS) 0755255B / American Heart Association (Southeast Affiliate); American Heart Association
- Language
- English
- Date published
- 02/01/2015
- Academic Unit
- Pulmonary, Critical Care, and Occupational Medicine; Internal Medicine
- Record Identifier
- 9984359802502771
Metrics
6 Record Views