Loss of AMPK action on double mutant S1157D/S1158D. Representative current records (top) and fully activated I/V relations (bottom) normalized to cell capacitance, from HEK293F cells in control conditions (black) and after AICAR treatment (red). While cells transfected with wild-type hHCN4 (left) show a standard response to AICAR treatment, cells transfected with S1157D/S1158D double-mutant channels in day-matched experiments (right) have a reduced basal current and are unresponsive to AICAR. Linear fitting of I/V curves yielded the following normalized conductance values (nS/pF): 1.20, 0.63 (left) and 0.68, 0.60 (right) for wild-type and AICAR-treated cells, respectively. Statistical analysis and data comparisons are shown in Tables 1 and S2.