Ling Wang1, Robert E W Fyffe, Luo Lu. 1. Division of Molecular Medicine, Harbor-UCLA Medical Center, The David Geffen School of Medicine, University of California Los Angeles, Torrance 90502, USA.
Abstract
PURPOSE: Voltage-gated K(+) channels maintain salt and water balance and normal function of corneal epithelial cells. To determine their identity, Kv channel types were sought in cultured rabbit corneal epithelial cells and in the intact rat corneal epithelium. METHODS: Immunohistochemistry and Western blot analysis were performed to detect K(+) channels in the membrane and cell lysates of rat and SV-40-transformed rabbit corneal epithelial (RCE) cells, using specific antibodies. The whole-cell patch clamp was used to characterize the biophysical and pharmacologic properties of the K(+) current in RCE cells. RESULTS: Expressions of K(+) channel types in corneal epithelial cells were detected by using a panel of specific anti-K(+) channel antibodies. Western blot analysis, using specific anti-K(+) channel antibodies including anti-Kv1.1, -2.1, -3.1, -3.2, -3.4, -4.2, and -4.3, demonstrated that in corneal epithelial cells Kv3.4 channel was highly expressed in whole-cell lysates and in cell membrane preparations. The anti-Kv3.4 channel antibody produced intense immunoreactivity in both RCE cells and rat corneal epithelium. Fluorescence immunostaining and avidin-biotin-peroxidase complex immunostaining confirmed localization of Kv3.4 channels in the cell membrane of both RCE and rat corneal epithelial cells. Voltage depolarization-activated K(+) currents in RCE cells were inhibited by applications of either 4-aminopyridine (4-AP, at micromolar levels), alpha-dendrotoxin at nanomolar levels, or blood-depressing substance-I at nanomolar levels. CONCLUSIONS: Biochemical and pharmacological profiles of the voltage-gated, 4-AP-sensitive K(+) channel in rat and RCE cells resemble characteristics of a Kv3.4 channel, a member of the Shaw subfamily. This channel may play important roles in maintaining normal function of corneal epithelium.
PURPOSE: Voltage-gated K(+) channels maintain salt and water balance and normal function of corneal epithelial cells. To determine their identity, Kv channel types were sought in cultured rabbit corneal epithelial cells and in the intact rat corneal epithelium. METHODS: Immunohistochemistry and Western blot analysis were performed to detect K(+) channels in the membrane and cell lysates of rat and SV-40-transformed rabbit corneal epithelial (RCE) cells, using specific antibodies. The whole-cell patch clamp was used to characterize the biophysical and pharmacologic properties of the K(+) current in RCE cells. RESULTS: Expressions of K(+) channel types in corneal epithelial cells were detected by using a panel of specific anti-K(+) channel antibodies. Western blot analysis, using specific anti-K(+) channel antibodies including anti-Kv1.1, -2.1, -3.1, -3.2, -3.4, -4.2, and -4.3, demonstrated that in corneal epithelial cells Kv3.4 channel was highly expressed in whole-cell lysates and in cell membrane preparations. The anti-Kv3.4 channel antibody produced intense immunoreactivity in both RCE cells and rat corneal epithelium. Fluorescence immunostaining and avidin-biotin-peroxidase complex immunostaining confirmed localization of Kv3.4 channels in the cell membrane of both RCE and rat corneal epithelial cells. Voltage depolarization-activated K(+) currents in RCE cells were inhibited by applications of either 4-aminopyridine (4-AP, at micromolar levels), alpha-dendrotoxin at nanomolar levels, or blood-depressing substance-I at nanomolar levels. CONCLUSIONS: Biochemical and pharmacological profiles of the voltage-gated, 4-AP-sensitive K(+) channel in rat and RCE cells resemble characteristics of a Kv3.4 channel, a member of the Shaw subfamily. This channel may play important roles in maintaining normal function of corneal epithelium.
Authors: John L Ubels; Rachel E Van Dyken; Julienne R Louters; Mark P Schotanus; Loren D Haarsma Journal: Exp Eye Res Date: 2011-03-04 Impact factor: 3.467
Authors: John L Ubels; Mark P Schotanus; Susan L Bardolph; Loren D Haarsma; Leah R Koetje; Julienne R Louters Journal: Exp Eye Res Date: 2009-10-28 Impact factor: 3.467