Ion channel imaging using pHluorin fragments and full length fluorescent ion channel proteins.
Ion channel function has been linked to disease states such as atrial fibrillation, supraventricular tachycardia, hypertrophic cardiomyopathy and hypertension, as well as headaches, the prevention of brain damage, and other disorders. Despite the clear links between ion channels and these diseases, there remains a need for an increased understanding of how these channels function and how they are regulated, which could lead to an increased understanding of these diseases and new targets to treat them. Researchers at the University of Michigan have developed ion channel constructs in which full length ion channel proteins were attached to fluorescent proteins or pHluorin fragments in order to study protein assembly, movement, localization, and regulation. These fluorescent constructs will be used as tools to understand how various molecules affect ion channel assembly and function, and provide a means for the development of new therapies directed at the regulation of ion channels.
Fluorescent Ion Channel Proteins for Immunohistochemistry, Immunocytochemistry, and Biomolecular Fluorescence Complementation.
Ion channels are proteins that regulate the movements of ions across the plasma membrane. Ion channel regulators are chemical or pharmacological agents that alter the function of these ion channels by increasing or restricting the movement of ions through the channel, or by increasing or decreasing the presence of the channel itself, either through biogenesis (creation and assembly of channel), degradation (destruction of the channel), or through internalization. Here Ion channels are attached to various fluorescent proteins, including enhanced and bright versions of fluorescent proteins. These fluorescently tagged proteins can be used to determine Ion channel biogenesis and degradation, trafficking to or from the membrane where they work, or recycling back to the plasma membrane once they have been internalized. These fluorescent constructs can be used in conjunction with immunocytochemistry and immunohistochemistry to detect changes in expression and localization under varying conditions and with high-through put screening, in the presence of test compounds, to identify compounds that are ion channel modulators. Further these fluorescent constructs can be used in conjunction with Biomolecular Fluorescence Complementation (BiFC) to visualize localization, assembly, and interactions of ion channels, and their individual subunits, with a higher special and temporal resolution than was previously attainable. These constructs can be used over time (minutes to lifetime of the protein), with temperature changes (4C to 40C), with pH changes (1 to 14), and in the presence of almost any compound. Understanding how these channels are assembled, regulated, and how they function, will provide insight into new ways to target ion channels with chemical therapies for the treatment of ion channel disorders.
Applications and Advantages
: - Direct visualization of ion channels.
High-through put screening.
Immunohistochemistry and Immunocytochemistry
Biomolecular Fluorescence Complementation to determine protein- protein and subunit-subunit interaction.
Advantages: - Enhanced and bright versions of fluorescent proteins allow for higher resolution. - High-through put screening makes drug discovery faster, easier, and more cost effective, with low reagent and sample volume.
-Real-time surface channel detection. -Visualization and detection of many ion channels at once due to multiple fluorophores.