A loudspeaker-driven system for rapid and multiple solution exchanges in patch-clamp experiments
Abstract
A new and inexpensive system allowing rapid and synchronized changes of solutions around a membrane patch or a cell under voltage-clamp conditions is described. Four plastic capillary tubings (OD 640 gin; ID 430 ~tm) were glued together horizontally and attached to a coil of a commercially available loudspeaker. Servo-control of the position of the coil allowed the mouth of any of the capillaries to be positioned near the pipette tip within 6 ms. A high flow speed of the test solution was crucial to achieve rapid solution exchange. At a flow speed of 5 cm/s, complete exchange of the external environment of a frog ventricular cell was achieved within 20-30 ms. The time course of solution change was found to be 3-5 times faster at the tip of an open patch pipette. To preserve the physical integrity of the cell, the cell was usually perfused by a control capillary at a slow velocity (0.2-0.4 cm/s) and test solutions flowing out of adjacent capillaries at high velocity (4-5 cm/s) were applied to the cell only for short periods. Determination of the three-dimensional contamination profile around the mouth of the control capillary allowed the optimal conditions for the use of the system to be established and possible sources of contamination to be avoided between adjacent capillaries with unmatched flow speeds. Successive and multiple changes in external solutions could be easily synchronized with voltage-clamp depolarizations to examine the time course of the effect of drugs on voltageoperated ion channels. An example of this application is given with rapid applications of the dihydropyridine agonist (-)BayK 8644 to the L-type Ca 2+ channel current in frog ventricular myocytes.
Domains
Life Sciences [q-bio]
Origin : Files produced by the author(s)