PURPOSE: To determine the presence of Kir2.1 channels in native bovine corneal endothelial (BCE) cells and assess their contribution to the resting membrane potential. METHODS: RT-PCR and Western blot analysis were used to detect the expression of Kir2.1 mRNA and protein in native BCE cells. Whole-cell patch-clamp recording was used to characterize Kir2.1 currents in freshly isolated, single BCE cells, as well as in BCE cell clusters. The contribution of Kir2.1 channels to the membrane potential (V(m)) was assessed by whole-cell recording in the zero-current clamp mode in the absence and presence of Ba(2+). RESULTS: RT-PCR analysis confirmed that Kir2.1 was expressed in the native BCE cells. Western blot analysis with native BCE cell protein and a polyclonal anti-Kir2.1 antibody revealed a approximately 60-kDa band that was blocked by the corresponding synthetic Kir2.1 peptide. Both single BCE cells and BCE cell clusters exhibited an inwardly rectifying K(+) (Kir) current that was dependent on the extracellular K(+) concentration. The Kir current was blocked by external Ba(2+) or Cs(+) in a voltage- and concentration-dependent manner. In 5 mM K(+) Ringer's, the V(m) of cell clusters averaged -40.0 +/- 4.1 mV (n = 14) and in 140 mM K(+) Ringer's it depolarized to -7.4 +/- 1.8 mV. Application of Ba(2+) in 5 mM K(+) Ringer's produced a concentration-dependent depolarization of V(m), with 10 mM Ba(2+) depolarizing V(m) from -53.4 +/- 4.8 mV to -27.8 +/- 6.3 mV (n = 6). CONCLUSIONS: Native BCE cells express functional Kir2.1 channels that help determine the membrane potential and probably also play a role in transendothelial transport.
PURPOSE: To determine the presence of Kir2.1 channels in native bovine corneal endothelial (BCE) cells and assess their contribution to the resting membrane potential. METHODS: RT-PCR and Western blot analysis were used to detect the expression of Kir2.1 mRNA and protein in native BCE cells. Whole-cell patch-clamp recording was used to characterize Kir2.1 currents in freshly isolated, single BCE cells, as well as in BCE cell clusters. The contribution of Kir2.1 channels to the membrane potential (V(m)) was assessed by whole-cell recording in the zero-current clamp mode in the absence and presence of Ba(2+). RESULTS: RT-PCR analysis confirmed that Kir2.1 was expressed in the native BCE cells. Western blot analysis with native BCE cell protein and a polyclonal anti-Kir2.1 antibody revealed a approximately 60-kDa band that was blocked by the corresponding synthetic Kir2.1 peptide. Both single BCE cells and BCE cell clusters exhibited an inwardly rectifying K(+) (Kir) current that was dependent on the extracellular K(+) concentration. The Kir current was blocked by external Ba(2+) or Cs(+) in a voltage- and concentration-dependent manner. In 5 mM K(+) Ringer's, the V(m) of cell clusters averaged -40.0 +/- 4.1 mV (n = 14) and in 140 mM K(+) Ringer's it depolarized to -7.4 +/- 1.8 mV. Application of Ba(2+) in 5 mM K(+) Ringer's produced a concentration-dependent depolarization of V(m), with 10 mM Ba(2+) depolarizing V(m) from -53.4 +/- 4.8 mV to -27.8 +/- 6.3 mV (n = 6). CONCLUSIONS: Native BCE cells express functional Kir2.1 channels that help determine the membrane potential and probably also play a role in transendothelial transport.
Authors: Sang Joon Ahn; Ibra S Fancher; Jing-Tan Bian; Chong Xu Zhang; Sarah Schwab; Robert Gaffin; Shane A Phillips; Irena Levitan Journal: J Physiol Date: 2016-12-26 Impact factor: 5.182
Authors: Victor G Romanenko; Yun Fang; Fitzroy Byfield; Alexander J Travis; Carol A Vandenberg; George H Rothblat; Irena Levitan Journal: Biophys J Date: 2004-10-01 Impact factor: 4.033