Tethered blockers as molecular 'tape measures' for a voltage-gated K⁺ channel

The propagation of electrical signals in excitable cells is orchestrated by a molecular family of voltage-dependent ion channel proteins. These K⁺, Na⁺, and Ca⁺⁺, channels are all composed of four identical or similar units, each containing six transmembrane segments (S1-S6) in a roughly four- fold symmetric structure. The S5-S6 sequences fold into a central pore unit, which is surrounded by a voltage-gating module composed of S1-S4. The recent structure of KcsA, a two-transmembrane bacterial K⁺ channel, illuminates the physical character of the pore unit, but little is known about the arrangement of the surrounding S1-S4 sequences. To locate regions of this gating module in space, we synthesized a series of compounds of varying length that function as molecular 'tape measures': quaternary ammonium (QA) pore blockers that can be tethered to specific test residues. We show that in a Shaker K⁺ channel, the extracellular ends of S1 and S3 are -30 Å from the tetraethylammonium (TEA) blocking site at the external opening of the pore. A portion of the S3-S4 loop is, at 17-18 Å, considerably closer.