Evidence for the interaction of endophilin a3 with endogenous Kca2.3 channels in PC12 cells.

BACKGROUND/AIMS: Small-conductance calcium-activated (SK) channels play an important role by controlling the after-hyperpolarization of excitable cells. The level of expression and density of these channels is an essential factor for controlling different cellular functions. Several studies showed a co-localization of K(Ca)2.3 channels and Endophilin A3 in different tissues. Endophilin A3 belongs to a family of BAR- and SH3 domain containing proteins that bind to dynamin and are involved in the process of vesicle scission in clathrin-mediated endocytosis. METHODS: Using the yeast two-hybrid system and the GST pull down assay we demonstrated that Endophilin A3 interacts with the N-terminal part of K(Ca)2.3 channels. In addition, we studied the impact of this interaction on channel activity by patch clamp measurements in PC12 cells expressing endogenous K(Ca)2.3 channels. K(Ca)2.3 currents were activated by using pipette solutions containing 1 µM free Ca(2+). RESULTS: Whole-cell measurements of PC12 cells transfected with Endophilin A3 showed a reduction of KCa2.3 specific Cs(+) currents indicating that the interaction of Endophilin A3 with K(Ca)2.3 channels also occurs in mammalian cells and that this interaction has functional consequences for current flowing through K(Ca)2.3 channels. Since K(Ca)2.3 specific currents could be increased in PC12 cells transfected with Endophilin A3 with DC-EBIO (30 µM), a known SK-channel activator, these data also implicate that Endophilin A3 did not significantly remove K(Ca)2.3 channels from the membrane but changed the sensitivity of the channels to Ca(2+) which could be overcome by DC-EBIO. CONCLUSION: This interaction seems to be important for the function of K(Ca)2.3 channels and might therefore play a significant role in situations where channel activation is pivotal for cellular function.

Verapamil- and state-dependent effect of 2-aminoethylmethanethiosulphonate (MTSEA) on hK(v)1.3 channels.

BACKGROUND AND PURPOSE: T-cells usually express voltage-gated K(v) 1.3 channels. These channels are distinguished by their typical C-type inactivation. Therefore, to be able to rationally design drugs specific for the C-type inactivation state that may have therapeutic value in autoimmune disease therapy, it is necessary to identify those amino acids that are accessible for drug binding in C-type inactivated channels. EXPERIMENTAL APPROACH: The influence of 2-aminoethylmethanethiosulphonate (MTSEA) on currents through wild-type human K(v)1.3 (hK(v)1.3) and three mutant channels, hK(v)1.3_L418C, hK(v)1.3_T419C and hK(v)1.3_I420C, in the closed, open and inactivated states was investigated by the patch-clamp technique. KEY RESULTS: Currents through hK(v)1.3_L418C and hK(v)1.3_T419C channels were irreversibly reduced after the external application of MTSEA in the open state but not in the inactivated and closed states. Currents through hK(v)1.3_I420C channels were irreversibly reduced in the open and inactivated states but not in the closed state. In the presence of verapamil, the MTSEA modification of hK(v)1.3_T419C and hK(v)1.3_I420C channels was prevented, while the MTSEA modification of hK(v)1.3_L418C channels was unaffected. CONCLUSION AND IMPLICATIONS: From our experiments, we conclude that the activation gate of all mutant channels must be open for modification by MTSEA and must also be open during inactivation. In addition, the relative movement of the S6 segments that occur during C-type inactivation includes a movement of the side chains of the amino acids at positions 418 and 419 away from the pore lining. Furthermore, the overlapping binding site for MTSEA and verapamil does not include position 418 in hK(v) 1.3 channels.