Super-resolution scanning patch clamp reveals clustering of functional ion channels in adult ventricular myocyte.

rationaleCompartmentation of ion channels on the cardiomyocyte surface is important for electric propagation and electromechanical coupling. The specialized T-tubule and costameric structures facilitate spatial coupling of various ion channels and receptors. Existing methods such as immunofluorescence and patch clamp techniques are limited in their ability to localize functional ion channels. As such, a correlation between channel protein location and channel function remains incomplete.

objectiveTo validate a method that permits routine imaging of the topography of a live cardiomyocyte and study clustering of functional ion channels from a specific microdomain. METHODS AND

resultsWe used scanning ion conductance microscopy and conventional cell-attached patch clamp with a software modification that allows controlled increase of pipette tip diameter. The sharp nanopipette used for topography scan was modified into a larger patch pipette that could be positioned with nanoscale precision to a specific site of interest (crest, groove, or T-tubules of cardiomyocytes) and sealed to the membrane for cell-attached recording of ion channels. Using this method, we significantly increased the probability of detecting activity of L-type calcium channels in the T-tubules of ventricular cardiomyocytes. We also demonstrated that active sodium channels do not distribute homogenously on the sarcolemma instead, they segregate into clusters of various densities, most crowded in the crest region, that are surrounded by areas virtually free of functional sodium channels.

conclusionsOur new method substantially increases the throughput of recording location-specific functional ion channels on the cardiomyocyte sarcolemma, thereby allowing characterization of ion channels in relation to the microdomain where they reside.