Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Olivet Martinez - Kansas State University
Co-Author(s): Peying Fong, Kansas State University, Manhattan, KS
SLC5A8 is a transmembrane protein which functions as a sodium-coupled monocarboxylate co-transporter and is a member of the SLC5 gene family of sodium-coupled cotransporters. The founding member of the SLC5 gene family is SLC5A1, which mediates cotransport of sodium and glucose (or galactose). In addition, previous research found SLC5A1 permeable to water. In this study, we hypothesize that SLC5A8 also mediates osmotic water transport. Due to their low intrinsic osmotic water permeability and well-studied capacity for heterologous expression, Xenopus laevis oocytes are an ideal experimental system for this study. To test the hypothesis, cRNA encoding human SLC5A8 (hSLC5A8) was expressed in Xenopus laevis oocytes (two groups: 5 ng of RNA/oocyte or 50 ng of RNA/oocyte). If hSLC5A8 permeates water, oocyte burst times subsequent to exposure to different hypotonic challenges would correlate with osmotic water entry. Oocytes injected with aquaporin-1 (AQP1), a water channel protein, served as positive controls. Negative controls comprised un-injected and water-injected oocytes. Test and control Xenopus laevis oocytes were incubated in buffered isotonic medium (OR-3) at 15 °C and measurements of burst times commenced 24 hours post-injection. After 24 hours, test and control oocytes were transferred from an isotonic solution to hypotonic solutions of varying osmolalities (isotonic: 214.4 mOsm/kg (100% tonicity); hypotonic: 118.4 mOsm/kg (55% tonicity), 86.4 mOsm/kg (40% tonicity), 22.4 mOsm/kg (10% tonicity)) over the course of 4 days and burst times were measured. Rupture of oocytes injected with hSLC5A8 was observed upon transfer to all hypotonic solutions tested. Oocytes injected with 5 ng of hSLC5A8 and challenged maximally with the 10% solution ruptured after 12.07 min (day 1), 25.98 min (day 2), and 28.52 min (day 3). Burst time decreased precipitously (3.02 min) on day 4, suggestive of cell death. Rupture times after maximal challenge of oocytes injected with 50 ng of hSLC5A8 decreased for 3 days post-injection (day 1: 9.6 min; day 2: 7.63 min; day 3: 0.7 min), but rebounded on day 4 (2.58 min). Burst time for oocytes injected with the positive control, AQP1, behaved as expected. Additionally, for both the test and positive control oocytes, average burst time decreased as osmolality levels decreased. We conclude human SLC5A8 can mediate osmotic water transport. Validation of this conclusion will require additional experimental analysis, including calculation of osmotic water permeability.
Funder Acknowledgement(s): This work was supported by National Science Foundation grant No. 1305059. This work was also supported by National Institutes of Health/National Institute of General Medical Sciences Award 1R15GM101674-01A1.
Faculty Advisor: Peying Fong, pfong@vet.ksu.edu
Role: Dr. Fong determined the project idea and the experimental system. While the oocytes were being collected and injected by the lab technician, I read literature on previous research. Dr. Fong and I agreed on a hypothesis. After oocyte preparation, I measured rupture times for each trial and oocyte condition. I transferred both control and experimental oocytes to different solutions with varying osmolalities and recorded their burst times. Dr. Fong and I analyzed the data collected and formed a conclusion.