Super-Resolution Scanning Patch-Clamp Reveals Clustering of Functional Ion Channels in the Adult Ventricular Myocyte
Rationale: Compartmentation of ion channels on the cardiomyocyte surface is important for electrical propagation and electromechanical coupling. The specialized T-tubule and costameric structures facilitate spatial coupling of various ion channels and receptors. Existing methods like 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.
Objective: To validate a method that permits to routinely image the topography of a live cardiomyocyte, and then study clustering of functional ion channels from a specific microdomain.
Methods and Results: We 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 which can 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 but rather, they segregate into clusters of various densities -most crowded in the crest region- that are surrounded by areas virtually free of functional sodium channels.
Conclusions: Our new method substantially increases the throughput of recording location-specific functional ion channels on the cardiomyocyte sarcolemma, thus allowing characterization of ion channels in relation to the microdomain in which they reside.
- scanning ion conductance microscopy
- cardiomyocyte structure
- calcium channel
- sodium channels
- ion channel
- Received November 1, 2012.
- Revision received February 25, 2013.
- Accepted February 25, 2013.