Different fluorescent labels report distinct components of spHCN channel voltage sensor movement

Abstract

Voltage clamp fluorometry was used to probe the S4 helix movement in the voltage sensing domain of the sea urchin HCN channel expressed in Xenopus oocytes. Markedly different fluorescence responses were obtained with either ALEXA-488 or MTS-TAMRA covalently linked to Cys332 at the N-terminal end of S4. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly showing characteristics consistent with it reporting the initial inward movement of S4 in agreement with previous studies. In contrast, MTS-TAMRA fluorescence was slower and correlated with the early phase of channel opening. In addition, a slow fluorescence component was resolved with both labels that tracked the development of the mode shift or channel hysteresis. This was quantitated as an increased deactivation tail current delay with concomitantly longer activation periods and was found to depend strongly on the presence of K+ ions in the pore. This indicated that the microenvironment of the fluorescent probes attached to Cys332 was strongly influenced by conformational changes in the pore domain. Collisional quenching experiments established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. This was supported by structural predictions based on homology modelling of spHCN in the closed and open conformations with covalently linked fluorophores. This study demonstrates that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal three distinct biophysical properties: voltage-sensor movement, early channel opening and mode-shift. These data support the use of different labelling probes to interrogate distinct biophysical aspects of voltage-gated membrane proteins.