Figure 1. Dimeric Kir2.1* constructs for the introduction of inner cavity cysteines. (A) Dimeric constructs were generated by fusing two copies of Kir2.1* using a 6X-glycine linker introduced by PCR. In all dimeric constructs, the front half comprises the background nonreactive Kir2.1* channels, and the back half comprises a cysteine-substituted Kir2.1* channel. (B) Steady-state spermine block was examined in 1 and 100 µM spermine by pulsing membrane voltage between −100 and +50 mV in 10-mV steps. Spermine block in Kir2.1* dimer channels is similar to spermine block in WT Kir2.1 and is unaffected by exposure to MTS reagents. Mean data are fit with a three-state model described in Materials and methods. (C) Use of dimeric cysteine constructs ensures modest current reduction in cysteine-substituted Kir2.1* dimers after exposure to MTSEA or MTSET, although modification by MTSET causes more dramatic current reduction at both positions examined (169C and 176).
Figure 1.

Dimeric Kir2.1* constructs for the introduction of inner cavity cysteines. (A) Dimeric constructs were generated by fusing two copies of Kir2.1* using a 6X-glycine linker introduced by PCR. In all dimeric constructs, the front half comprises the background nonreactive Kir2.1* channels, and the back half comprises a cysteine-substituted Kir2.1* channel. (B) Steady-state spermine block was examined in 1 and 100 µM spermine by pulsing membrane voltage between −100 and +50 mV in 10-mV steps. Spermine block in Kir2.1* dimer channels is similar to spermine block in WT Kir2.1 and is unaffected by exposure to MTS reagents. Mean data are fit with a three-state model described in Materials and methods. (C) Use of dimeric cysteine constructs ensures modest current reduction in cysteine-substituted Kir2.1* dimers after exposure to MTSEA or MTSET, although modification by MTSET causes more dramatic current reduction at both positions examined (169C and 176).

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