Figure 6.
Functional analysis of the GsuK channel. (A) 86Rb+ flux assays of GsuK-containing liposomes in the presence of various nucleotides. Data are averages of two measurements and normalized against the control sample without nucleotide. (B)–(D) Single channel traces and I–V curves of the wild-type channel and L97D mutant. Currents were recorded using giant liposome patch clamping with 150 mM NaCl and 150 mM KCl in the pipette and bath solutions, respectively. (E) Sample traces of wild-type channel in the presence and absence of intracellular Ca2+ (left) and the plot of [Ca2+]-dependent single channel open probability of wild-type GsuK and L97D mutant (right). Both pipette and bath solutions contain symmetrical 150 mM KCl. Data for wild-type channel are fitted to the Hill equation with K1/2 of 197 uM and Hill coefficient n = 2.3. Data are mean ± SEM of seven measurements. (F) Sample traces of partially deactivated GsuK channel in the presence and absence of 2 mM ADP. Shown on the right is the plot of [Ca2+]-dependent single channel open probability of wild-type GsuK in the presence of 2 mM various adenine-containing nucleotide. Both pipette and bath solutions contain symmetrical 150 mM KCl. Data are fitted to the Hill equation with K1/2 = 210 μM and n = 2 for AMP, K1/2 = 350 μM and n = 1.4 for ADP, and K1/2 = 370 μM and n = 1.3 for NAD+. Data are mean ± SEM of five measurements.