Third party funded individual grant
Start date : 01.01.2014
End date : 31.12.2015
Activation of skeletal muscle requires a delicate cascade of ion channel function and electro-chemical signaling. Only even spread of excitation into t-tubules ensures synchronized activation of force. The distribution of ion channels in the t-system thus has to be finely tuned to account for ion concentration changes in diffusion restricted spaces. This becomes even more relevant in chronic muscle diseases that are associated with substantial remodeling of the organ but also cellular remodeling where sarcomeres and triads can be sterically deranged or uncoupled. We aim to determine the 3D distribution of ion channels in the t-system alongside with subcellular architecture using multiphoton immuno-fluorescence and Second-harmonic generation microscopy.
During the DAAD funding period, we will adress the following work packages:
1. Perform electrophysiology recordings in single muscle fibres of wt and mdx edl muscle to record either Kir2.1 currents or CIC-1 mediated Cl- currents. From the recordings over a whole voltage range, I-V curves will be reconstructed. The recordings will be attempted in resting muscle and muscle that has been undergoing a strenuous fatigue protocol using bouts of tetanic activations.
2. Perform immuno-fluorescence (IF) labeling of Kir2.1 or CIC-1 channels in permeabilzed single fibres. We aim to use the same single fibres as for (1) following the electrophysiology procedures and additional fibres to increase statistics. IF will be performed on a multiphoton microscope and the imaging performed through 0.3 µm sections within the single cell. The 3D aspect of the ion channel localization will be obtained from 3D rendering. To correlate ion channel localizations to tubular membranes, in some cases, t-membranes will be also stained using di-8ANEPPS.
3. Additionally to (2), simultaneous SHG recordings will be performed to label-free visualize the sarcomeric architecture in normal and mdx muscle. From the IF and SHG signal reconstruction, we can correlate ultrastructure with ion channel morphology and establish corresponding channel maps.