The Kir2.3 potassium channel possesses cysteines at extracellular and intracellular positions where cysteine-binding reagents, such as thimerosal, are known to act (Bannister et al. 1999). Additionally it possesses a cysteine half-way down the pore-lining M2 region at position C160, although it is not known if this residue is accessible to extracellularly applied thimerosal. The object of this work was to investigate the accessibility to thimerosal of this residue.

Extracellularly applied thimerosal is already known to act on extracellularly facing cysteines C79 and C140, so that experiments were carried out in the background of the channel C79S/C140S. Mutations were made using the PCR Quickchange method, and the further mutation C160S was made. RNA for these mutants and wild-type Kir2.3 was injected into Xenopus oocytes and two-electrode voltage-clamp recordings were made 1-2 days later at room temperature. Hyperpolarising pulses were applied at 0.1 Hz from a holding potential of 0 mV. The effects of bath-applied thimerosal (100 mM) were obtained by perfusing continuously during repetitive pulses to -100 mV.

The current kinetics and I-V curves for the mutants were very similar to those of wild-type. Thimerosal induced an inhibition of the currents for the wild-type and all the mutants, but the extent of inhibition differed. The extent of inhibition by thimerosal of the triple mutant C79S/C140S/C160S (by 43.7 ± 2.0 %, n = 6, mean ± S.E.M.) was significantly (P < 0.05, Student’s unpaired t test) less than the inhibition of the double mutant C79S/C140S (by 84.5 ± 4.3 %, n = 5) or the wild-type channel (by 88.7 ± 3.2 %, n = 6). By comparing experiments where thimerosal was applied for 5 min in the presence or absence of stimulation, the effects of thimerosal were similar whether for wild-type or mutants, indicating lack of use-dependent effect of thimerosal. Furthermore, the inhibition induced by thimerosal was irreversible by washing for 5 min, but was reversed by the application of the reducing agent dithiothreitol, confirming that the effects occurred via covalent modification.

The results show that extracellularly applied thimerosal can reach residue C160 and produce inhibition. This M2 residue is therefore pore-facing and probably lies in an intracellular-facing large cavity, as also occurs for the corresponding residue in Kv2.1 (Lu et al. 2001) and in Kv6.2 (Loussouarn et al. 2000). Thimerosal is membrane permeable, and probably reaches this residue by first crossing the membrane and entering the cavity from the intracellular side.

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