Unraveling the kinetics and pharmacology of human PepT1 using solid supported membrane-based electrophysiology.

The human Peptide Transporter 1 (hPepT1) is known for its broad substrate specificity and its ability to transport (pro-)drugs. Here, we present an in-depth comprehensive study of hPepT1 and its interactions with various substrates via solid supported membrane-based electrophysiology (SSME). Using hPepT1-containing vesicles, we could not identify any peptide induced pre-steady-state currents, indicating that the recorded peak currents reflect steady-state transport.

A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development.

The lysosomal cation channel TMEM175 is a Parkinson's disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings.

Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation.

Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining K and K for different sugars, k values for sugar-induced conformational transitions and the effects of Na, Li, H and Cl on sugar binding and transport.

SSM-based electrophysiology, a label-free real-time method reveals sugar binding & transport events in SGLT1.

Here, we present a solid-supported membrane (SSM)-based electrophysiological approach to study sugar binding and Na/glucose cotransport by SGLT1 in membrane vesicles. SSM-based electrophysiology delivers a cumulative real-time current readout from numerous SGLT1 proteins simultaneously using a gold-coated sensor chip. In contrast to conventional techniques, which mainly operate with voltage steps, currents are triggered by sugar or sodium addition.

Functional Characterization of SLC Transporters Using Solid Supported Membranes.

Here, we present a protocol for the functional characterization of the H-coupled human peptide transporter PepT1 and sufficient notes to transfer the protocol to the Na-coupled sugar transporter SGLT1, the organic cation transporter OCT2, the Na/Ca exchanger NCX, and the neuronal glutamate transporter EAAT3.The assay was developed for the commercially available SURFER N1 instrument (Nanion Technologies GmbH) which applies solid supported membrane (SSM)-based electrophysiology. This technique is widely used for the functional characterization of membrane transporters with more than 100 different transporters characterized so far.

SSM-Based Electrophysiology for Transporter Research.

Functional characterization of transport proteins using conventional electrophysiology can be challenging, especially for low turnover transporters or transporters from bacteria and intracellular compartments. Solid-supported membrane (SSM)-based electrophysiology is a sensitive and cell-free assay technique for the characterization of electrogenic membrane proteins. Purified proteins reconstituted into proteoliposomes or membrane vesicles from cell culture or native tissues are adsorbed to the sensor holding an SSM.

Discovery of Compounds that Positively Modulate the High Affinity Choline Transporter.

Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the gene).

Electrophysiological characterization of the archaeal transporter NCX_Mj using solid supported membrane technology.

Sodium-calcium exchangers (NCXs) are membrane transporters that play an important role in Ca(2+) homeostasis and Ca(2+) signaling. The recent crystal structure of NCX_Mj, a member of the NCX family from the archaebacterium Methanococcus jannaschii, provided insight into the atomistic details of sodium-calcium exchange. Here, we extend these findings by providing detailed functional data on purified NCX_Mj using solid supported membrane (SSM)-based electrophysiology, a powerful but unexploited tool for functional studies of electrogenic transporter proteins.

KCNMA1 encoded cardiac BK channels afford protection against ischemia-reperfusion injury.

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells.

TRPML3.

TRPML3 belongs to the MCOLN (TRPML) subfamily of transient receptor potential (TRP) channels comprising three genes in mammals. Since the discovery of the pain sensing, capsaicin- and heat-activated vanilloid receptor (TRPV1), TRP channels have been found to be involved in regulating almost all kinds of our sensory modalities. Thus, TRP channel members are sensitive to heat or cold; they are involved in pain or osmosensation, vision, hearing, or taste sensation.

Studying mechanosensitive ion channels with an automated patch clamp.

Patch clamp electrophysiology is the main technique to study mechanosensitive ion channels (MSCs), however, conventional patch clamping is laborious and success and output depends on the skills of the operator. Even though automated patch systems solve these problems for other ion channels, they could not be applied to MSCs. Here, we report on activation and single channel analysis of a bacterial mechanosensitive ion channel using an automated patch clamp system.