Figure 1.
(A) a bright field image of cobblestone-like cells. Scale bar = 100 µm. (B) Gpr39 mRNA levels were evaluated by Taqman gene expression analysis in both muscle tissues and cultured cells. After 7 days in culture, Gpr39 levels were up-regulated 163.9 fold in the cultures (n = 3). (C) Cells were loaded with Fura2-AM. Shown are ratiometric images of Ca2+ signals before and after 100 µM Zn2+ challenge. [Ca2+]i is indicated on a rainbow scale with blue representing low [Ca2+]i and orange/red high [Ca2+]i. (D) Ca2+ signals were elicited in cells cultured from wild-type mice, but not Gpr39−/− mice. (E) Zn2+ induces Ca2+ signals in Ca2+ free bath solution. (F) Cells were pre-incubated with or without 1 µM thapsigargin to deplete intracellular Ca2+ stores. Thapsigargin treatment not only abolished Zn2+ elicited Ca2+ signals, but also resulted in a decrease in [Ca2+]i after Zn2+ application, the mechanism of which is still unknown.
Figure 2.
Correlation between GPR39 expression and function in the culture cells.
(A) the Zn2+ induced Ca2+ signals were measured in the cultured cells after 1, 3 and 4 days in vitro. (B) a bar chart summarizing the total Ca2+ response and percentage of Zn2+ responsive cells in Panel A. (C) shows the expression of Gpr39 mRNA in the corresponding cultures over the same culturing periods.
Figure 3.
GPR39 expressing cells are fibroblast-like cells.
(A-C) Representative images for immuno-labelling of C-Kit, CD34, PDGFRα and SK3 in cultured cobblestone-like cells. Scale bar = 50 µm. (D-F) the mRNA expression levels of CD34 and pdgfra in the cultures increased 6.87±0.96 (p<0.01) and 3.39±1.55 (p>0.05) fold respectively, while c-kit expression decreased 0.0013±0.0003 (p<0.05) fold.
Figure 4.
Zn2+ induces a large current in FLCs.
(A) Membrane potentials were recorded in current clamp configuration. Zn2+ induced rapid membrane depolarization in FLCs cultured from wild-type mice, but not Gpr39−/− cells. (B) Bar chart summarizing Zn2+ induced membrane depolarization in FLCs as in panel A. p<0.01. (C and D) Zn2+ induced a large current in wild-type. Current was sampled at +80 and −80 mV. (C) shows a time-series plot for a typical Zn2+-induced current in FLCs (n = 21). (D) shows the corresponding I-V relationship. (E and F) are similar experiments using FLCs isolated from GPR39 knockout mice and show that no Zn2+-induced current was observed in Gpr39−/− FLCs (n = 11).
Figure 5.
Zn2+ induced currents are carried by chloride channels.
(A) Zn2+ induced current when potassium channels were blocked by CsCl in the pipette solution, excluding a major role of K+ channels (n = 5). (B) Zn2+ still induced a current when recordings were conducted in symmetrical NMDG-Cl solutions, suggesting chloride channels are involved (n = 4). (C) CFTR antagonist did not have any effect on Zn2+-induced currents (n = 3). (D) Time courses of the current measured at +80 mV from individual cells. Zn2+-induced currents decreased gradually after reaching peak amplitude (top). Application of a TMEM16A antagonist resulted in a rapid blockade of this current (bottom) (n = 7), suggesting TMEM16A is involved. (E) Expression of TMEM16A in FLCs was detected by reverse transcription PCR using gene specific primers. NAC stands for no amplification control.
Figure 6.
GPR39 activation is functionally linked with TMEM16A channel opening.
(A) Time courses for the current measured at +80 mV from individual cells. Both Ionomycin (1 µM) and Zn2+ evoked currents in FLCs from WT mice (n = 11, top trace), while Zn2+ was ineffective in Gpr39−/− FLCs (n = 6, bottom trace). The data are summarized in panel (B). (C) Knockdown of Tmem16a by siRNA in FLCs were verified by Taqman PCR (94% reduction in Tmem16a expression), whereas Gpr39 expression were unaffected (n = 3). (D) Zn2+-induced Ca2+ responses were measured in FLCs. No difference was observed between control and Tmem16a siRNA transfected cells (n = 54 and 67). In contrast, the size of Zn2+-induced current was reduced to about 32% in transfected cells (n = 7 and 12).