vamg6792@uxa.cso.uiuc.edu (Vincent A Mazzarella) (05/08/91)
The following is a one page synopsis of a recent one-hour seminar. The impressions are my own and do not reflect accuracy of the facts contained in the presentation. Corrections and discussion are welcomed. Perforated Patch -- Patch Clamping Whole cell recording using a patch pipette technique has several problems. Calcium can diffuse out of the cytoplasm through the ruptured membrane at the site of the pipette attachment into the pipette medium, which often contains the calcium chelator EGTA. Because of this and the washout of other molecules, cellular currents disappear with time. Furthermore, with a non-intact cell membrane, receptor-channel coupling cascades may not be seen if the intermediates are washed out. In the perforated patch, the cell membrane between the walls of the pipette is not ruptured as in the whole cell recording. Instead, the fungicide nystatin is added to the pipette solution. This agent causes 8 A channels to form which will pass molecules with MW < 200 but will not pass multivalent ions. Thus Na+, K+, Cl-, and Cs+ are ions that can pass. As the channels are made in the membrane, the resistance of the seal decreases from 80 MOhm to 20 MOhm (100 ug/ml nystatin). In lacrimal cells, ACh stimulates muscarinic receptors that through IP3 and Ca++ open K+ channels. In whole cell patches, ACh stimulated current diminishes with time and repeated stimulations, disappearing within 15 min. of patching. However, perforated patches show no current diminution with time (i.e. repeated stimuli). Similar results are seen for Ca++ currents in pituitary GH3 cells, and for Ca++-dep't Cl- currents in ACTH secreting, CRF sensitive ATT20 cells of the pituitary. Using the perforated patch technique, the role of Ca++ in this latter cell line was studied. One question addressed was the removal of Ca++. When extracellular Na+ is replaced with TMA or TEA, there is a prolonged Ca++, as assayed by a prolonged Cl- current. This implicates that the 3:1 Na+/Ca++ exhanger is important in removing calcium. This was substantiated by noting that the tail currents were longer and of greater duration under these conditions and when TEA was rapidly substituted for Na+ (using a movable pipet on a solenoid mount) shortly after CRF stimulation caused the current onset. To see whether the Na+/H+ exhanger actually played an intermediate role, the latter experiment was repeated in varying the external pH using 80 mM HEPES from 7.3 to 8.0. No difference at different pHs was seen, so the Na/H exhanger does not appear to play a role. To see if pH affects the Ca pump (Na/Ca pump?) the intracellular Calcium was measured using the fluorescent indicator FURA-2 and a photomultiplier tube. Fluorescence after a CRF stimulation showed a prolonged Ca++ level when pH was raised from 7.3 to 8.0 or to below 6.8. The removal of calcium was prolonged at the extreme pHs even when a smaller CRF stimulus was administered. Thus pH seems to affect the Ca++ pump. Rich Kramer was mentioned for work measuring single channel currents using a perforated, excised outside out patch, called a "perforated vesicle." This is desirable for the same reason that perforated patch whole cell recordings are desirable. From a one-hour seminar in Spring 1990, at the Univ. of Illinois, Urbana-Champaign. -- Vincent Mazzarella College of Medicine, Neuroscience Program University of Illinois, Urbana-Champaign e-mail: mazz@vmd.cso.uiuc.edu