A Llobet1, X Gasull, J Palés, E Martí, A Gual. 1. Laboratori de Neurofisiologia, Departament de Ciències Fisiològiques I-Institut d'Investigacions Biomèdiques August Pi i Sunyer, Facultat de Medicina, Universitat de Barcelona, Spain.
Abstract
PURPOSE: To study the presence of inwardly rectifying K(+) (Kir) channels in cultured bovine (BTM) and human (HTM) trabecular meshwork cells. METHODS: Cultures of BTM and HTM cells were obtained by an extracellular matrix digestion technique. Whole-cell patch-clamp recordings of BTM cells were performed with the appropriate solutions to detect K(+) currents. Also, Western blot analysis of Kir2.1 protein expression was performed on both cultured BTM and HTM cells. RESULTS: A strong inwardly rectifying current at negative potentials to the equilibrium potential for K(+) (E(K+)) and highly selective for K(+) was detected in 60% of cultured BTM cells. The slope conductance of the inward rectification was more pronounced when the extracellular [K(+)] was increased and was proportional to [K(+)](0.45). The current was blocked by Ba(2+) and Cs(+) in a voltage- and concentration-dependent manner, with K(d) at 0 mV, of 74.7 microM and 45.6 mM, respectively. Current amplitude was reduced by increasing extracellular [Ca(2+)]. The current was insensitive to 10 microM glibenclamide and 10 nM tertiapin. The application of 100 microM 8-Br-cAMP reduced the current by 50%. Kir2.1 channel expression was detected in confluent monolayers of BTM and HTM cells by Western blot analysis. CONCLUSIONS: A population of cultured BTM cells expressed an inwardly rectifying K(+) current that illustrates the biophysical and pharmacologic characteristics of the detected Kir2.1 channel protein. Kir2.1 channels are also thought to be present in HTM cells. Kir2.1 channels could be related to TM physiology, because they are involved in contractile and cell volume regulatory responses, two mechanisms that modify TM permeability.
PURPOSE: To study the presence of inwardly rectifying K(+) (Kir) channels in cultured bovine (BTM) and human (HTM) trabecular meshwork cells. METHODS: Cultures of BTM and HTM cells were obtained by an extracellular matrix digestion technique. Whole-cell patch-clamp recordings of BTM cells were performed with the appropriate solutions to detect K(+) currents. Also, Western blot analysis of Kir2.1 protein expression was performed on both cultured BTM and HTM cells. RESULTS: A strong inwardly rectifying current at negative potentials to the equilibrium potential for K(+) (E(K+)) and highly selective for K(+) was detected in 60% of cultured BTM cells. The slope conductance of the inward rectification was more pronounced when the extracellular [K(+)] was increased and was proportional to [K(+)](0.45). The current was blocked by Ba(2+) and Cs(+) in a voltage- and concentration-dependent manner, with K(d) at 0 mV, of 74.7 microM and 45.6 mM, respectively. Current amplitude was reduced by increasing extracellular [Ca(2+)]. The current was insensitive to 10 microM glibenclamide and 10 nM tertiapin. The application of 100 microM 8-Br-cAMP reduced the current by 50%. Kir2.1 channel expression was detected in confluent monolayers of BTM and HTM cells by Western blot analysis. CONCLUSIONS: A population of cultured BTM cells expressed an inwardly rectifying K(+) current that illustrates the biophysical and pharmacologic characteristics of the detected Kir2.1 channel protein. Kir2.1 channels are also thought to be present in HTM cells. Kir2.1 channels could be related to TM physiology, because they are involved in contractile and cell volume regulatory responses, two mechanisms that modify TM permeability.
Authors: Oleg Yarishkin; Tam T T Phuong; Colin A Bretz; Kenneth W Olsen; Jackson M Baumann; Monika Lakk; Alan Crandall; Catherine Heurteaux; Mary E Hartnett; David Križaj Journal: J Gen Physiol Date: 2018-11-16 Impact factor: 4.086