Repetitive depletion and recovery of intracellular K+ in retinal Müller glial cells during whole-cell voltage-clamp.
A procedure was developed allowing repetitive depletion and recovery of K+ from the cell interior of Müller glial cells without patch pipette perfusion. To this end the whole-cell voltage-clamp technique using tight seal pipettes was applied to enzymatically isolated Müller cells of the guinea pig. When K+ was replaced by Cs+ and/or NMDG in the pipette solution voltage-activated outward currents could be generated similar to those found with normal K(+)-containing intracellular solution. This was the result of the distribution of K+ across the cell membrane due to the negative holding potential, i.e., K+ accumulated at the intracellular side of the cell membrane. This distribution should be favored by the low input resistance of Müller cells. K+ could also be removed from the cell interior by depolarizing voltage steps. Simultaneously, K+ influx had to be cut off by using a K(+)-free extracellular solution or blocking K+ channels using Ba2+. This procedure lead to reduction and eventually cessation of the K+ outward currents, indicating extinction of the intracellular K+ pool. The process is reversible, i.e., outward currents can be evoked again by depolarizing voltage steps after renewed application of extracellular K+ and/or removal of Ba2+. Hence, the intracellular K+ pool is filled up again. The described method was applied to demonstrate the dependence of the Müller cell Na+/glutamate transporter on the intracellular K+ concentration.
Francke
M
M
Paul-Flechsig-Institute for Brain Research, Department of Neurophysiology, Leipzig, Germany.
Pannicke
T
T
Reichelt
W
W
D000818:Animals; D001464:Barium / Q000494:pharmacology; D006168:Guinea Pigs; D009457:Neuroglia / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods*; D011188:Potassium / Q000494:pharmacology; D015221:Potassium Channels / Q000502:physiology*; D012160:Retina / Q000502:physiology*
D015221:Potassium Channels; D001464:Barium; D011188:Potassium