Human oocytes express ATP-sensitive K(+) channels

Abstract

Background: ATP-sensitive K(+) (K(ATP)) channels link intracellular metabolism with membrane excitability and play crucial roles in cellular physiology and protection. The K(ATP) channel protein complex is composed of pore forming, Kir6.x (Kir6.1 or Kir6.2) and regulatory, SURx (SUR2A, SUR2B or SUR1), subunits that associate in different combinations. The objective of this study was to determine whether mammalian oocytes (human, bovine, porcine) express K(ATP) channels.

Methods: Supernumerary human oocytes at different stages of maturation were obtained from patients undergoing assisted conception treatments. Bovine and porcine oocytes in the germinal vesicle (GV) stage were obtained by aspirating antral follicles from abattoir-derived ovaries. The presence of mRNA for K(ATP) channel subunits was determined using real-time RT-PCR with primers specific for Kir6.2, Kir6.1, SUR1, SUR2A and SUR2B. To assess whether functional K(ATP) channels are present in human oocytes, traditional and perforated patch whole cell electrophysiology and immunoprecipitation/western blotting were used.

Results: Real-time PCR revealed that mRNA for Kir6.1, Kir6.2, SUR2A and SUR2B, but not SUR1, were present in human oocytes of different stages. Only SUR2B and Kir6.2 mRNAs were detected in GV stage bovine and porcine oocytes. Immunoprecipitation with SUR2 antibody and western blotting with Kir6.1 antibody identified bands corresponding to these subunits in human oocytes. In human oocytes, 2,4-dinitrophenol (400 µM), a metabolic inhibitor known to decrease intracellular ATP and activate K(ATP) channels, increased whole cell K(+) current. On the other hand, K(+) current induced by low intracellular ATP was inhibited by extracellular glibenclamide (30 µM), an oral antidiabetic known to block the opening of K(ATP) channels.

Conclusions: In conclusion, mammalian oocytes express K(ATP) channels. This opens a new avenue of research into the complex relationship between metabolism and membrane excitability in oocytes under different conditions, including conception.

Figure 1

K_ATP channel subunits mRNAs in human oocytes. (A) Original progress curves for the real-time PCR amplification of SUR1, SUR2A, SUR2B, Kir6.1 and Kir6.2 cDNA in human oocytes. Curves labelled with subscripts C and E correspond to control and oocyte curves respectively. (B and C) Bar graphs showing cycle threshold for the real-time PCR amplification of SUR1, SUR2A, SUR2B, Kir6.1 and Kir6.2 from human oocytes (B) and human skeletal muscle (C) that served as a positive control. Absence of a bar pattern in graphs that is depicted in symbols means that no PCR product was obtained for this particular gene.

Figure 2

K_ATP channel subunits mRNAs in bovine oocytes. Bar graphs showing cycle threshold for the real-time PCR amplification of SUR1, SUR2A, SUR2B, Kir6.1 and Kir6.2 from bovine oocytes (A) and H9C2 cells (B) that served as a positive control. Absence of a bar pattern in graphs that are depicted in symbols means that no PCR product was obtained for this particular gene.

Figure 3

K_ATP channel subunits mRNAs in porcine oocytes. Bar graphs showing cycle threshold for the real-time PCR amplification of SUR1, SUR2A, SUR2B, Kir6.1 and Kir6.2 from porcine oocytes (A) and H9C2 cells (B) that served as a positive control. Absence of a bar pattern in graphs that are depicted in symbols means that no PCR product was obtained for this particular gene.

Figure 4

Kir6.1 protein that physically associates with SUR2A and/or SUR2B in human oocytes. A western blot of anti-SUR2 immunoprecipitate from human oocytes and A549 cells (cells that do not express K_ATP channels natively) with anti-Kir6.1 antibody under depicted conditions. The arrow indicates a signal corresponding to Kir6.1.

Figure 5

Whole cell K⁺ current in human oocytes in response to procedures that alter intracellular ATP levels. (A) Original superimposed membrane currents in a human oocyte in the absence (control) and presence (DNP) of DNP (400 µM), and corresponding I–V relationship (left graph) and DNP-sensitive component of the current (right graph; DNP-sensitive component of the current was obtained by digital subtraction of the current in the presence and absence of DNP). (A1) Bar graph showing whole cell current at 80 mV in the absence and presence of DNP (400 µM). Bars are mean ± SEM (n = 4). *P < 0.05. (B) Original superimposed membrane currents in a human oocyte in the absence (low ATP) and presence (glybenclamide) of glybenclamide (30 µM), and corresponding I–V relationship (left graph) and glybenclamide-sensitive component of the current (right graph; glybenclamide-sensitive component of the current was obtained by digital subtraction of the current in the absence and presence of glybenclamide). (B1) Bar graph showing whole cell current at 80 mV in the absence and presence of glybenclamide (30 µM). Bars are mean ± SEM (n = 3). *P < 0.05.

Figure 6

Cartoon summarizing possible structure, regulation and function of K_ATP channels in human oocytes based on the findings from the present study as well as findings from previous studies that have investigated K_ATP channels in other cell types and the physiology of oocytes (Carrasco et al., 2001; Crawford et al., 2002a,b; Tosti and Boni, 2004; Jovanović et al., 2005, 2009a,b; Nichols, 2006; Van Blerkom et al., 2008; Van Blerkom, 2009).