Pore- and state-dependent cadmium block of I_Ks channels formed with MinK-55C and wild-type KCNQ1 subunits

Human MinK and KCNQ1 subunits assemble to form I_Ks channels. When MinK position 55 is mutated to cysteine (MinK-55C), I_Ks channels can be blocked by external cadmium. (Cd²⁺). We have supported a pore-associated location for MinK-55C because Cd²⁺ block is sensitive to voltage, permeant ions on the opposite side of the membrane (trans-ions), and external tetraethylammonium (TEA), an I_Ks pore-blocker. Two recent reports argue that MinK-55C is distant from the pore: one finds TEA does not affect Cd²⁺ block if channels are formed with a KCNQ1 mutant (K318I, V319Y) that increases TEA affinity; the second proposes that Cd²⁺ binds between MinK-55C and a cysteine in KCNQ1 that is posited to lie toward the channel periphery. Here, these discrepancies are considered. First, Cd²⁺ block of MinK-55C channels formed with wild-type KCNQ1 is shown to depend not only on voltage and trans-ions but state (showing decreased on-rate with increased open time and blocker trapping on channel closure). Conversely, MinK-55C channels with K318I, V319Y KCNQ1 are found to demonstrate Cd²⁺ block that is independent of voltage, trans-ions and state (and to have a lower unitary conductance): thus, the KCNQ1 mutations alter the process under study, yielding Cd²⁺ inhibition that is pore-independent and, perforce, TEA-insensitive. Second, MinK-55C channels are found to remain sensitive to Cd²⁺ despite mutation of any single native cysteine in KCNQ1 or all nine simultaneously; this suggests no KCNQ1 cysteine binds Cd²⁺ and can serve to localize MinK-55C. Despite many concerns that are enumerated, we remain obliged to conclude that Cd²⁺ enters and leaves the pore to reach MinK-55C, placing that residue in or near the pore.