Figure 4.

Encoding of the noncanonical amino acid citrulline quantifies the importance of a positive charge in an otherwise minimally modified side chain at position 230. (A) Structure of arginine and citrulline. (B) Cartoon representing encoding of citrulline into KCNQ3-R230 channels. Co-injection of pyrrolysine tRNA-citrulline with KCNQ3 cRNA containing the site-directed stop codon UAG (nonsense codon) at 230 into Xenopus oocytes (top panel). After 2 d of expression, citrulline (red) was incorporated into KCNQ3 channels at position 230 (bottom panel and left zoom in cartoon). (C) Extrapolated tail conductance from KCNQ3-A315T-R230Citrulline (red inset) was normalized (see Materials and methods) and plotted versus test voltages (G(V), red solid line; means ± SEM; n = 7). Normalized G(V) of KCNQ3-A315T (WT Q3, dotted black line) and KCNQ3-A315T-R230C (R230C, solid black line) are shown for comparison. Lines represent the fitted theoretical voltage dependencies (see Materials and methods, Eqs. 1 and 2). Inset shows a representative current trace from KCNQ3-R230Citrulline (red). Dotted line represents zero current. Cells were held at −80 mV and stepped to potentials between −160 mV and +60 mV in 10-mV intervals for 0.5 s, followed by a step to −40 mV to record tail currents.