Figure 3.

A negatively charged residue at position 50 is critical for regulation by Ca²⁺. (A) Hemichannel currents from oocytes expressing D50C mutant hemichannels in low and high extracellular Ca²⁺ to assess the effects of chemical modification with MTS reagents. Currents were elicited by a pulse to 0 mV from a holding potential of −80 mV before (black trace) or in the presence of MTSES⁻ (red traces) or MTSET⁺ (blue traces). (B) Holding currents obtained at −80 mV for oocytes expressing D50C mutant hemichannels incubated in low (0.25 mM) or high (10 mM) external Ca²⁺. The addition of MTSES⁻ increased the holding current in low extracellular Ca²⁺ (top trace) and decreased the holding current in the presence of high Ca²⁺ (bottom trace). (C) Deactivation time constants for D50C mutant hemichannels at different Ca²⁺ concentrations before (dotted line; from Fig. S3) and after chemical modification with MTSES⁻ (red triangles) or MTSET⁺ (blue triangles). Time constants obtained after MTSES⁻ modification coincide with the solid line (from Fig. 1 C) that corresponds with the linear fit for wild-type hemichannels. Conversely, the time constant obtained after MTSET⁺ overlaps with the linear fit for D50C mutants with no modification. The data represent mean ± SEM of at least three independent measurements.