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Cell membranes are highly intricate systems comprising numerous lipid species and membrane proteins, where channel proteins, lipid molecules, and lipid bilayers, as continuous elastic fabric, collectively engage in multi-modal interplays. Owing to the complexity of the native cell membrane, studying the elementary processes of channel-membrane interactions necessitates a bottom-up approach starting from forming simplified synthetic membranes. This is the rationale for establishing an in vitro membrane reconstitution system consisting of a lipid bilayer with a defined lipid composition and a channel molecule. Recent technological advancements have facilitated the development of asymmetric membranes, and the contact bubble bilayer (CBB) method allows single-channel current recordings under arbitrary lipid compositions in asymmetric bilayers. Here, we present an experimental protocol for the formation of asymmetric membranes using the CBB method. The KcsA potassium channel is a prototypical model channel with huge structural and functional information and thus serves as a reporter of membrane actions on the embedded channels. We demonstrate specific interactions of anionic lipids in the inner leaflet. Considering that the local lipid composition varies steadily in cell membranes, we `present a novel lipid perfusion technique that allows rapidly changing the lipid composition while monitoring the single-channel behavior. Finally, we demonstrate a leaflet perfusion method for modifying the composition of individual leaflets. These techniques with custom synthetic membranes allow for variable experiments, providing crucial insights into channel-membrane interplay in cell membranes., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In the almost four decades since its inception, the patch clamp technique has transitioned from a specialist skill to a method commonly used among many others in a lab. Development of patch clamp instrumentation has not been steady: A boost of product releases in rapid succession by multiple manufacturers in the 1990s had slowed to a trickle by the mid-2000s. In 2016, Sutter Instrument's entry into the market of turnkey patch clamp amplifier systems, defined as an amplifier with matching data acquisition hardware and software, caused a fresh breeze in a field in danger of going stale. Sutter has meanwhile completed the product line, culminating in the flagship dPatch ® Ultra-fast, Low-noise Digital Amplifier. The dPatch System constitutes a contemporary, digital design that features many firsts, including digital signal compensation, an extremely high bandwidth and fully integrated dynamic clamp capability, paired with the increasingly popular SutterPatch ® Software.This chapter compares feature sets of the new Sutter instrumentation with the established platforms by the other two providers of turnkey systems, Axon Instruments by Molecular Devices and HEKA Elektronik by Harvard Bioscience. A variety of products from other manufacturers, who rely on combination with components from other sources rather than offering turnkey systems, are listed, but for their conceptual diversity not compared at a great level of detail. The chapter further covers architectural considerations for patch clamp systems, headstage design, data acquisition strategies and efficient structuring of the recorded data, controlling and monitoring periphery, advanced technologies, such as software lock-in amplifier capability and dynamic clamp features, and application modules for efficient analysis of action potentials and postsynaptic events.

Many enzymatic activity assays are based on either (1) identifying and quantifying the enzyme with methods such as western blot or enzyme-linked substrate assay (ELISA) or (2) quantifying the enzymatic reaction by monitoring the changing levels of either product or substrate. We have generated an outer membrane protein G (OmpG)-based nanopore approach to distinguish enzyme identity as well as analyze the enzyme's catalytic activity. Here, we engineered an OmpG nanopore with a peptide cut site inserted into one of its loops to detect proteolytic behavior. In addition, we generated an OmpG nanopore with a single-stranded DNA attached to a loop for analyzing nucleolytic cleavage. This OmpG nanopore approach may be highly useful in analyzing specific enzymes in complex biological samples, or in directly determining kinetics of enzyme-substrate complex association and dissociation.

Electrophysiology is an essential tool aiding the study of the functions and dysfunctions of electrically excitable cells and their networks. The patch clamp method is a refined electrophysiological technique that can directly measure the membrane potential and/or the amount of current passing across the cell membrane. The patch clamp technique is also incredibly versatile and can be used in a variety of different configurations to study a range of properties, from spontaneous cell firing activity in native tissue to the activation and/or deactivation kinetics of individual channels expressed in recombinant cell lines. In this chapter we give an overview of patch clamping and how the different configurations can be set up and applied to electrophysiological research.