KcsA channels have been extensively studied to understand K+ permeation through ion channels. It has been postulated that the opening of the conducting pore at the bundle crossing of the four TM2 helices has to occur before ion conduction. It remains unclear whether this process happens in one cooperative step, as suggested for voltage-gated channels, or if the subunits can act independently. Here, we present a study of the subunits' cooperativity using single channel fluorescence spectroscopy. We have shown previously (Blunck et al., 2006) that we can monitor the movement of the TM2. This is done by labeling them at the C-terminus of TM2 with an environment sensitive dye. We introduced purified and labeled KcsA channels into supported lipid bilayer and imaged their fluorescence time course. Fluctuations in the fluorescence intensity prior to photobleaching were interpreted as the movement of the four subunits. The associated open probability followed the KcsA pH dependence, and mean values similar to our results in planar lipid bilayer were observed. We observed occurrence of simultaneous gating of all four subunits, but also independent gating of single subunits. We analyzed the time traces with a hidden Markov model, considering photobleaching and coupling energy between subunits, in order to quantify the coupling energy. We found a coupling energy with a mean value of about 2.5 kT, which seems to be slightly higher at low pH. Current results showed occurrence of subconductance levels which we hypothesize to be correlated with the partial opening of the lower gate. Therefore, we are currently working on simultaneously detecting current and fluorescence of a single KcsA channel in a planar lipid bilayer.