Abstract
Intracellular ion channels are involved in multiple signaling processes, including such crucial ones as regulation of cellular motility and fate. With 95% of the cellular membrane belonging to intracellular organelles, it is hard to overestimate the importance of intracellular ion channels. Multiple studies have been performed on these channels over the years, however, a unified approach allowing not only to characterize their activity but also to study their regulation by partner proteins, analogous to the patch clamp “golden standard”, is lacking. Here, we present a universal approach that combines the extraction of intracellular membrane fractions with the preparation of patchable substrates that allows to characterize these channels in endogenous protein environment and to study their regulation by partner proteins. We validate this method by characterizing activity of multiple intracellular ion channels localized to different organelles and by providing detailed electrophysiological characterization of the regulation of IP3R activity by endogenous Bcl-2. Thus, after synthesis and reshaping of the well-established approaches, organelle membrane derived patch clamp provides the means to assess ion channels from arbitrary cellular membranes at the single channel level.
Highlights
Localized ion channels are known to play important role in various cellular signaling pathways which are often directly involved in the regulation of cellular fate
A series of much earlier publications, dating back 20–30 years, some even prior to the advent of the classical patch clamp, hinted at approaches that potentially could resolve the above issues: collecting different membrane fractions followed by fusion of this material with sufficiently large liposomes prepared via some pre-established routines, such as enlarging small unilamellar vesicles (SUVs) by subjecting them to freeze-thaw or dehydration-rehydration cycles[13,14], or by inducing blister formation in lipids deposited directly on the chamber floor[15]
The technique employs as a first step the isolation of either a specific membrane fraction or a collection of cell membranes that are intended for further functional characterization
Summary
Localized ion channels are known to play important role in various cellular signaling pathways which are often directly involved in the regulation of cellular fate. The publications that report on electrophysiological properties of these channels often utilize planar lipid bilayer experiments This well established technique has certain limitations: large electrical capacitance of the bilayer resulting in low acquisition bandwidth and the nessesity of purification of the investigated channel protein, compromising the study of regulation of ion channels by their native protein partners[9,10]. The relatively tedious manipulations involved and the need for two independent micro-manipulators instead of one prevented, a wide adoption of this approach Summing up, these prior publications presented interesting protocols and encouraged the study of the intracellular channel activity, often requiring tedious manipulations, mirrored by author’s own experience[18,19]. A native IP3R channel in nuclear envelope typically exhibits drastically different gating properties, with significantly higher Popen than that of the purified protein reconstituted in a planar lipid bilayer[10]. Rigorous study of the arbitrarily localized intracellular ion channels and their regulation by partner proteins (such as in the case of the IP3R s and Bcl-2 in ER and MAMs; see above) is still beyond the scope of the approaches readily available to broad scientific community
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