Muscarinic activation of ionic currents measured by a new whole-cell recording method.

TLDR

The study introduces a nystatin‑permeabilized patch method to prevent wash‑out of muscarinic responses in rat lacrimal gland cells. The method uses nystatin‑permeabilized cell‑attached patches to maintain cytoplasmic integrity while allowing electrical access. Using the nystatin method, muscarinic activation of Ca‑activated K currents persisted for at least one hour, allowed dose‑response curves with saturation above ~1 µM ACh, and showed a concentration‑dependent reduction in activation delay to 0.7–1.0 s.

Abstract

A new method is described as an alternative to whole-cell recording in order to prevent "wash-out" of the muscarinic response to acetylcholine (ACh) in rat lacrimal gland cells. The membrane of a cell-attached patch is permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without the loss or alteration of cytoplasmic compounds necessary for the maintenance of the response to ACh. With normal whole-cell recording in these cells, the response to ACh, seen as the activation of Ca-activated K and Cl currents, lasts for approximately 5 min. With the nystatin method, the response is not diminished after 1 h. Nystatin, applied extracellularly, is shown to cause a rapid and reversible increase of membrane conductance to cations. In the absence of wash-out, we were able to obtain dose-response curves for the effect of ACh on Ca-activated K currents. An increase of [ACh] caused an increase in the K current, with apparent saturation at concentrations above approximately 1 microM ACh. The delay between ACh application and the activation of K current was inversely related to [ACh] and reached a minimum value of 0.7-1.0 s at high [ACh].