The nanobody Nb.C1 potentiates human acid-sensing ion channel 1a and 1b

of activation, whereas at hASIC1b it shifts the pH-dependence of activation to more alkaline values by 0.38 pH units. Fusion of Nb.C1 to the ASIC1a-inhibiting peptide psalmotoxin 1 (PcTx1) generates a bivalent molecule, Nb.C1-PcTx1, which retains PcTx1's mechanism of inhibition at hASIC1a but exhibits markedly prolonged activity consistent with increased avidity. At hASIC1b, Nb.C1-PcTx1 produces a large 1.12 pH unit alkaline shift in the pH dependence of activation and sustained inward currents near physiological pH with slow washout. Together, these results demonstrate that Nb.C1 acts as a nanomolar potentiator of both human ASIC1a and ASIC1b and that fusion to PcTx1 markedly prolongs ligand activity through avidity, resulting in persistent off-target modulation at ASIC1b.