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. 2011 Mar 17;286(18):16414–16425. doi: 10.1074/jbc.M110.198010

FIGURE 8.

FIGURE 8.

Identification of a possible CORM-2 binding site in the Kv2.1 channel. A, docking of a putative degradation product of CORM-2, Ru(DMSO)2(CO)2Cl2 (black), with Kv1.2 in the open and closed states is shown. The S3 and S4 are shown in red, the S4-S5 linker is in orange, and the pore domain of the adjacent subunit is in light yellow. Residues Val-261 (S3 segment, Leu-264 in Kv2.1), Gly-318 (S4-S5 linker, Gly-317 in Kv2.1), and Ala-397 (S6, Ala-396 in Kv2.1), whose substitution resulted in functional channels, are shown in blue. Residues Ser-324 (S5, Ser-323 in Kv2.1), Gly-313 (S4-S5 linker, Gly-313 in Kv2.1), Leu-328 (S5, Leu-327 in Kv2.1), and Gly-338 (S5, Gly-337 in Kv2.1), when mutated resulted in non-functional channels, are shown in purple. Position Gly-318 is marked with an asterisk. B, representative currents of eYFP-Kv2.1 and eYFP-Kv2.1G317V were activated by voltage pulses from −100 to 100 mV in 20-mV increments and deactivated at −120 mV. C, normalized G-V relations for the different Kv2.1 constructs in saline are shown. The G317V curve was normalized to the open probability measured from non-stationary noise analysis at 100 mV. Curves are fits to Equation 1 with following parameters: eYFP-Kv2.1, V1/2 = −33.2 ± 7.0 mV, z = 2.59 ± 0.22 e0; L264M, V1/2 = −28.3 ± 5.6 mV, z = 3.05 ± 0.60 e0; A396V, V1/2 = −37.1 ± 4.0 mV, z = 2.84 ± 0.30 e0, (n = 4). The continuous red curve is a representative G-V-relation of G317V in the presence of 0.7 mm CORM-2 obtained from a voltage ramp from −100 to 60 mV with 0.48 mV/s normalized to a fit of a single-barrier permeation model as described in supplemental Fig. 3D. D, CORM-2 dose-response curves for WT and mutant Kv2.1 channels at 100 mV are shown. Symbols are as in C. Continuous and dotted curves are fits to the Hill equation with following parameters: eYFP-Kv2.1, K1/2 = 0.56 mm, s = 1.91; L264M, K1/2 = 0.59 mm, s = 2.41; A396V, K1/2 = 0.63 mm, s = 3.67; n = 3–4. The G317V data were fit to the following product of two Hill equations: Icorm/Isal = 1 + Imax[(1 + [CORM-2]/K1/2,1)s1(1 + K1/2,2/[CORM-2])s2]−1 and obtained with the following parameters: Imax = 28, K1/2,1 = 0.73 mm, s1 = 4.0, K1/2,2 = 0.92 mm, s2 = 2.65 (n = 3–6). E, time course of Kv2.1 inhibition by 0.7 mm CORM-2 in the open (black symbols) and the closed (white symbols) state is shown. The first point in the curve marks the beginning of CORM-2 application. Red curves are fits to an exponential function with rates: kopen-state = 0.217 ± 0.041 s−1, kclosed-state = 0.207 s−1 (n = 6). F, shown is the time course of 0.5 mm CORM-2-action on G317V in the open (black symbols) and closed states (white symbols). Red curves are fits to an exponential with parameters: kopen-state = 0.045 ± 0.009 s−1, kclosed-state = 0.0625 s−1 (n = 3–6).