Measuring Extracellular Ion Gradients from Single Channels with Ion-Selective Microelectrodes

Abstract

Under many different conditions activated plasma membrane ion channels give rise to changes in the extracellular concentration of the permeant ion(s). The magnitude and duration of these changes are dependent on the electrochemical driving force(s) on the permeant ion(s) as well as conductance, open time, and channel density. We have modeled the change in the extracellular [K+] due to efflux through Ca2+-activated K+ channels, mSlo, to determine the range of parameters that would give rise to measurable signals in the surrounding media. Subsequently we have used extracellular, K+-selective microelectrodes to monitor localized changes in [K+]ext due to efflux through mSlo channels expressed in Xenopus oocytes. The rapid changes in [K+] show a close fit with the predicted model when the time response of the ion-selective microelectrode is taken into account, providing proof of the concept. Measurement of the change in extracellular ion concentration with ion-selective microelectrodes provides a noninvasive means for functional mapping of channel location and density, as well as characterizing the properties of ion channels in the plasma membrane.

Windows to cell function and dysfunction: Signatures written in the boundary layers

“…4 answers this question and illustrates the recording of single potassium channels. (33,34) Using the CHO heterologous system we also demonstrated that the microelectrode acts as an ion-trap, amplifying the signal from the channel. Obtaining a rapid response potas-sium electrode did require design changes in both the electrode body and the ionophore cocktail.…”

“…(28,29) Potentiometric applications have taken advantage of the several excellent ionselective cocktails available commercially, notably for hydrogen, potassium and calcium, with modified cocktails being developed specifically for self-referencing detection of chloride or sodium (32) and rapid response time potassium sensors. (33,34) Amperometric detection depends on reduction/oxidation chemistry. Detection of nitric oxide, oxygen and hydrogen peroxide are now routine applications (sensor fabrication (29,35) ).…”

Fast Response, Noninvasive, Potentiometric Microelectrodes Resolve Single Potassium Channel Activity in the Diffusive Boundary Layer of a Single Cell

“…The recent use of this design was able to resolve the transient changes (1 -2 s duration) of Ca 2þ and H þ in the extracellular spaces of the brain [8 -10]. We have employed the use of shorter columns to capture rapid K þ changes due to efflux through single Ca 2þ -activated K þ channels [11,12].…”

“…Microelectrodes were constructed with short columns (ca. 9 mm) of a mechanically stable K þ increases of 1 -10 mM and fit a diffusion model when response time and ion trapping by the ISM are considered [11,12]. In Figure 3A and B the rapid transients appear as spikes.…”

“…In order to extract the single channel characteristics we performed best-fit modeling of single events [11,12] and performed manual measurement and calculation of mean open time and open probability. While the modeling supported the fact that we were measuring extracellular K þ changes from single channels it was tedious.…”