In this chapter, a detailed protocol is given for ion-channel reconstitution in the two most used model membranes: planar bilayers and liposomes. In the planar bilayer section, methods are described for the expression of ion channels in Xenopus laevis oocytes, the isolation of their membranes, the insertion of ion channels into the bilayer by vesicle fusion, and the recording of single-ion channel current measurements at a constant applied voltage. The reconstitution of bacterial channels in liposomes is also given. It includes the expression and purification of bacterial channels in E. Coli host strain XL1-blue, the insertion of the channels in liposomes, and the recording of their currents by patch clamping.
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http://dx.doi.org/10.1007/978-1-59745-519-0_38 | DOI Listing |
In the human heart, the binding of cyclic adenosine monophosphate (cAMP), a second messenger, to hyperpolarization and cyclic nucleotide-gated (HCN) regulates the automaticity of pacemaker cells. Recent single-molecule binding studies show that cAMP bound to each subunit of purified tetrameric HCN channels independently, in contrast to findings in cells. To explore the lipid membrane's role in cAMP regulation, we reconstituted purified human HCN channels in various lipid nanodiscs and resolved single molecule ligand-binding dynamics.
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December 2024
Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA.
Maintaining a balance of inorganic phosphate (Pi) is vital for cellular functionality due to Pi's essential role in numerous biological processes. Proper phosphate levels are managed through Pi import and export, facilitated by specific Pi transport proteins. Although the mechanisms of Pi import have been extensively studied, the processes governing Pi export remain less understood.
View Article and Find Full Text PDFJ Physiol
December 2024
Section on Cellular Communication, Eunice Kennedy Shiver National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA.
The Drosophila neuromuscular junction (NMJ) is a powerful genetic system that has revealed numerous conserved mechanisms for synapse development and homeostasis. The fly NMJ uses glutamate as the excitatory neurotransmitter and relies on kainate-type glutamate receptors and their auxiliary protein Neto for synapse assembly and function. However, despite decades of study, the reconstitution of NMJ glutamate receptors using heterologous systems has been achieved only recently, and there are no reports on the gating properties for the recombinant receptors.
View Article and Find Full Text PDFBio Protoc
November 2024
Department of Biology, University of Padova, Padova, Italy.
The planar lipid bilayer (PLB) technique represents a highly effective method for the study of membrane protein properties in a controlled environment. The PLB method was employed to investigate the role of mitochondrial inner membrane protein 17 (MPV17), whose mutations are associated with a hepatocerebral form of mitochondrial DNA depletion syndrome (MDS). This protocol presents a comprehensive, step-by-step guide to the assembly and utilization of a PLB system.
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November 2024
Department of Chemistry, Mississippi State University, Starkville, MS, 39759, USA.
Voltage-gated sodium channels (Na) are complex macromolecular proteins that are responsible for the initial upstroke of an action potential in excitable cells. Appropriate function is necessary for many physiological processes such as heartbeat, voluntary muscle contraction, nerve conduction, and neurological function. Dysfunction can have life-threatening consequences.
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