&p.1:Abstract Members of the rat brain Kv1 family of cloned potassium channels are structurally highly homologous, but have diverse conductance and pharmacological char- acteristics. Here we present data on the effects of mutat- ing residues K533 in the P-region and H471 in the S4-S5 linker of Kv1.4 to their equivalent residues in Kv1.1 and Kv1.6 on single-channel conductance and sensitivity to external tetraethylammonium cations (TEA + ) and internal Mg 2+ . Exchange of residue K533 for its equivalent resi- due (Y) in Kv1.1 and Kv1.6 increased the single-channel conductance at both negative and positive potentials. This mutation is known to reduce the IC 50 for external TEA + from > 100 mM to 0.6 mM, almost identical to that for Kv1.1 (0.53 mM). We have now found that the additional exchange of residue H471 for the equivalent residue (K) in Kv1.6 increased the IC 50 for external TEA + from 0.6 mM (Kv1.4K533Y) to 2.39 mM; this is very close to that for wild-type Kv1.6 channels (2.84 mM). The muta- tion H471K alone was ineffective. We thus provide evi- dence that the S4-S5 linker does contribute to the chan- nel’s inner-pore region. Data on the block of Kv1 chan- nels by internal Mg 2+ indicate that while the binding site is probably situated within the deep-pore region, its exact location may be channel specific. &kwd:Key words Potassium channel · Single channel · Xenopus oocyte · Tetraethylammonium · Magnesium&bdy: Introduction Voltage-gated potassium (Kv) channels may be formed by the assembly of four α-subunits in homo- or hetero- oligomeric complexes. Hydropathy and model-building studies have predicted a common α-subunit topology of five hydrophobic membrane-spanning segments (S1-3, S5 and S6), one membrane-spanning segment containing a number of regularly spaced, positively charged amino acid side-chains (S4) and an additional shorter segment linking segments S5 and S6 and incorporating the P- or H5-region. The P-region is presumed to enter and exit the membrane from the extracellular side. The S4-S5 linker, the P-region and the S6 segment have, as a result of domain-swapping and mutational studies, all been im- plicated in the formation of the pore structure [5, 7, 12, 13, 16–18, 22–24]. Members of the Shaker-related Kv1 family of Kv α- subunits cloned from rat brain have pore-forming do- mains which differ in their primary sequence by only a few amino-acid residues, yet they give rise to channels which have diverse conductance and pharmacological profiles, as well as quite different gating characteristics, when expressed in Xenopus oocytes or mammalian cell- lines [4, 20]. The present study concerns Kv1.1 (RCK1) and Kv1.6 (RCK2), which give rise to delayed rectifier- type currents, and Kv1.4 (RCK4) which gives rise to fast- inactivating A-type currents. The single-channel conduc- tance of Kv1.4 is much smaller than that of either Kv1.1 or Kv1.6. In addition, Kv1.4 channels are relatively in- sensitive to external tetraethylammonium cations (TEA + ). The primary structure of all three channels is identical in the S6 region. There are 11 non-conserved residues in the S5-S6 linker, of which only three involve a difference in charge (Kv1.4 residues 507, 509 and 533; the P-region extending from P514 to P534), and only two non-con- served residues in the S4-S5 linker (Kv1.4 residues 471 J. M. Gòmez-Hernandez · L. A. Pardo · W. Stühmer MPI für Experimentelle Medizin, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany A. A. Elliott ( ✉ ) · S. H. Heinemann Max-Planck-Gesellschaft, AG Molekulare und zelluläre Biophysik, Drackendorfer Str. 1, D-07747 Jena, Germany C. Lorra · O. Pongs ZMNH Institut für neurale Signalverarbeitung, D-20246 Hamburg, Germany C. Lorra Institut für Neurobiologie, INF 364, D-69120 Heidelberg, Germany A. A. Elliott Department of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, UK&/fn-block: Pflügers Arch – Eur J Physiol (1997) 434:661–668 © Springer-Verlag 1997 ORIGINAL ARTICLE &roles:Juan M. Gòmez-Hernandez · Christoph Lorra Luis A. Pardo · Walter Stühmer · Olaf Pongs Stefan H. Heinemann · Alison A. Elliott Molecular basis for different pore properties of potassium channels from the rat brain Kv1 gene family &misc:Received: 25 February 1997 / Received after revision: 4 June 1997 / Accepted: 10 June 1997