Combined Single-Channel and Macroscopic Recording Techniques to Analyze Gating Mechanisms of the Large Conductance Ca2+ and Voltage Activated (BK) Potassium Channel

Abstract

Ion channels are integral membrane proteins that regulate membrane potentials and signaling of cells in response to various stimuli. The patch-clamp technique enables the study of single channels or a population of channels. The macroscopic recording approaches are powerful in revealing population-averaged behaviors of channels both under basal conditions and in response to various stimuli, modulators and drugs. On their own, however, these approaches can be insufficient for determinations of channel gating mechanisms as they do not accurately report channel open probabilities below 10(-2) to 10(-3). This obstacle can be overcome with the use of single-channel recording techniques. Single-channel recording techniques can be applied to one or a few channels to estimate P o over a larger range than macroscopic recordings. The combination of heterologous overexpression of ion channels with macroscopic and single-channel recordings can be applied to hundreds of channels to estimate P o between 1 and 10(-8). Here, we describe practical approaches of single-channel recordings that our laboratory utilizes. We also provide examples where the combined macroscopic and single channel approach can be employed to study gating mechanisms of the BK type, large conductance, Ca(2+) and voltage activated potassium channel in a mammalian expression system. The techniques presented should be generally applicable to the studies of ion channels in heterologous expression systems.

Downregulation of KCNMB4 expression and changes in BK channel subtype in hippocampal granule neurons following seizure activity

“…The numbers of channels in the patch was revealed by using a +120 mV voltage command to evoke near-maximal opening of channels (in 60 μM free Ca 2+ internal solution). Single channel data was idealized as outlined in [ 19 ]. Data is represented as averages ± S.E.M.…”