(A) Western blot of proteins located in the plasma membrane of X. laevis oocytes heterologously expressing the indicated CALHM channels. Protein was isolated after surface-biotinylation by binding to avidin-resin. The proteins are detected with specific antibodies recognizing the respective CALHM paralog. Left, CALHM2, center CALHM4, right CALHM6. The blot demonstrates the targeting of all three paralogs to the plasma membrane. Bands corresponding to the respective CALHM paralogs are indicated by asterisk. (B) Electrophysiological characterization of X. laevis oocytes heterologously expressing the paralogs CALHM1, CALHM2, CALHM4 and CALHM6 in comparison to control oocytes (neg.) recorded at extracellular solutions either containing 3 mM Ca2+ (3) or 0.5 mM EDTA and 0.5 mM EGTA (Ca2+-free, 0). Data show currents of individual oocytes (circle) recorded by two-electrode voltage-clamp (TEVC) at 60 (light blue) and −60 mV (light red). In each case, currents were tabulated at the end of a 5 s voltage step. Averages are shown as bars in red (−60 mV) and blue (60 mV), respectively. Dashed lines indicate mean current levels of control oocytes (neg.) recorded in Ca2+-free extracellular solutions at 60 and −60 mV. Currents measured for all investigated channels, except CALHM1, are not significantly different from control oocytes (as judged by a Student t-test). (C), Rectification of steady-state currents of oocytes displayed in (B) expressed as I60mV/I-60mV calculated for individual oocytes at 3 mM Ca2+ (3) and in Ca2+-free solutions (0) and averaged. The large value of CALHM1 reflects the activation of the protein at positive voltage in presence of Ca2+. (D) Ca2+-dependence of activation. Change of steady-state currents of oocytes displayed in (B) after Ca2+-removal expressed as InoCa2+/ICa2+ calculated from individual oocytes at −60 mV (n) and 60 mV (p) and averaged. The large value of CALHM1 at −60 mV reflects the strong activation of currents at negative voltages upon Ca2+-depletion. C, D, The difference between the corresponding values of the CALHM paralogs 2, 4, and 6 and neg. are statistically insignificant (as judged by a Student t-test). B-D, Data show averages of 27 (neg.), 21 (CALHM1), 26 (CALHM2), 19 (CALHM4) and 21 (CALHM6) oocytes respectively. Errors are standard deviations.