Visualizing ion channel dynamics at the plasma membrane

Abstract

Cardiac ion channels are surprisingly dynamic in nature, and are continuously formed, trafficked to specific subregions of plasma membrane, inserted in the plasma membrane, and removed to be degraded or recycled. Because of these movements, which affect channel availability, ion channel function is dependent on not just channel biophysical properties but channel trafficking as well. The development of molecular techniques to tag proteins of interest with fluorescent and other genetically encoded proteins, and of advanced imaging modalities such as total internal reflection microscopy (TIRF), have created new opportunities to understand the intracellular movement of proteins near the plasma membrane and their dynamics therein. In this article we present approaches for ion channel biologists to the use of fluorescent and nonfluorescent fusion proteins, techniques for cloning and expression of fusion proteins in mammalian cells, and imaging techniques for live-cell high-resolution microscopy. For illustration, original data are presented on creation of a stable cell line capable of inducible expression of connexin 43 tagged to green fluorescent protein and its distribution viewed with both wide-field epifluorescence and TIRF microscopy. With revolutionary advances in fluorescence microscopy, ion channel biologists are now entering a new realm of studying channel function, which is to understand the mechanisms of channel trafficking.

Figure 1. Doxycycline-induced expression of connexin43-EGFP in a stable cell line

HeLa cells expressing the rtTA-Advanced transactivator protein (Clontech) were transfected with pTRE-tight tetracycline-responsive vector encoding connexin43-EGFP and a linear selection marker (Clontech). The schematic above image panels illustrates that doxycycline is required for the transactivator protein rtTA to bind to the tetracycline-responsive element (TRE) preceding the promoter and gene of interest, allowing transcription to commence. Cells were incubated with (right panel) or without (left panel) 1.0 µg/ml Doxycycline (Sigma) for 24 h prior to fixation and the addition of nuclear counterstain DAPI (blue). Image acquisition was undertaken using a 60X objective.

Figure 2. Widefield Epifluorescence and TIRF microscopy

Widefield epifluorescence excites the entire cell being images, activating all fluorophores within the field of view. TIRF microscopy limits excitation depth to 10–100 nm activating only those fluorophores in and just below the plasma membrane. Images are of the same induced conexin43-EGFP expressing cell as viewed with widefield epifluorescence (left) and TIRF (right).