Localizzazione funzionale dei canali pacemaker : identificazione molecolare dei canali f nel nodo senoatriale di coniglio : l'interazione proteina-proteina localizza i canali f nei microdomini caveolari e ne modula le proprieta cinetiche e l'espressione in membrana

Growing evidence indicates that caveolae, specialized membrane microdomains, are important regulators of ion channels. An important function of caveolae is to cluster related proteins in close proximity to facilitate functional interactions. We have previously shown that pacemaker f-channels are localized in caveolin-enriched membrane fractions (Barbuti et al., 2004, Circ Res 94, 1325) and co-immunoprecipitate with the caveolar protein caveolin-3 in sinoatrial tissue. Since a caveolin-interacting sequence is found in the N-terminus of all HCN channel isoforms (WIIHPYSDF), we hypothesized that mutation of this consensus site in the HCN4 isoform, the most abundant in the sinoatrial node, would cause changes of channel properties similar to those caused by caveolar disorganization. We expressed rabbit HCN4 in CHO cells and used various techniques (point mutation, patch clamp, western blot, immunofluorescence) to verify this hypothesis. We mutated tyrosine 259 into a serine (Y259S). The Y259S channels had a V1/2 potential 24.6 mV more positive than that of the WT: -81 ± 2 mV n = 31; Y259S: -56.4 ± 1 mV n = 35). Also, analysis of time constants (tau) showed the presence of a significant rightward shift of activation/deactivation tau curves of mutated channels. Furthermore, the Y259S current density (-14.5 ± 2 pA/pF; n = 43) was significantly lower than WT current density (-53.5 ± 7.1 pA/pF; n = 41) at -125 mV. These data agree with immunofluorescence data showing a weaker HCN4 signal in CHO cells expressing mutated vs WT channels. Interestingly, the Y259S channels localized, like WT channels, to caveolin-enriched membrane fractions and co-immunoprecipitated with caveolin 1. In conclusion, our data show that the Y259S mutation causes changes of HCN4 properties qualitatively similar to, but quantitatively larger than, those reported for caveolae disorganization without affecting channel localization; furthermore this mutation causes a significant reduction of membrane channel density.