Adjacently and diagonally arranged Kv1.1 and 1.2 tandem-linked gene constructs gave channels that were distinguished by TEA, but not AgTX1, TsTX-Kα, or KTX. (A) Current traces recorded using QPatch from an adjacent (Kv1.1-1.1-1.2-1.2) and diagonal (Kv1.2-1.1-1.2-1.1) channel in the absence (black) and presence of TEA, AgTX1, TsTX-Kα, or KTX (blue). (B) Dose–response curves for the forward and reverse adjacent channels show an ∼10-fold lower affinity for TEA than the corresponding diagonals. ○, Kv1.1-1.1-1.2-1.2; n = 5 cells for each concentration, manual patch clamp; Hill equation fit IC₅₀ = 9.6 mM, slope = 0.8. •, Kv1.2-1.2-1.1-1.1; Qpatch, n = 2–3; 8.2 mM, 0.6. □, Kv1.1-1.2-1.1-1.2; n = 4, Qpatch; 0.9 mM, 0.6. (blue) □, Kv1.1-1.2-1.1-1.2; n = 6–8, manual patch clamp; 1.1 mM, 0.6. ▪, Kv1.2-1.1-1.2-1.1; n = 3–5, Qpatch; 0.8 mM, 0.7. (Inset) Non-concatenated (red ▴; n = 6–7; 0.4 mM, 0.6) and concatenated (black ▴; n = 2–4; 0.7 mM, 0.7) homotetrameric Kv1.1 channels showed almost identical sensitivity to TEA, as with the less sensitive homotetrameric Kv1.2 (red ▾; n = 2–4; 41 mM, 0.6) channel and the tandem-linked homotetramer (black ▾; n = 3; 36 mM, 0.5). All homomer data were from Qpatch. (C) Channel dimers showed TEA susceptibilities similar to that for the pair of adjacently arranged tetrameric concatamers. □, Kv1.1-1.2; n = 8; 9.3 mM, 1.0. •, Kv1.2-1.1; n = 5–6; 7.8 mM, 0.7. ◆, Kv1.1-1.2 + Kv1.2-1.1; n = 9–11; 9.5 mM, 0.4. Data were from Qpatch. Error bars represent ± SEM.