Potassium channels share a highly conserved stretch of eight amino acids, a K+ channel signature sequence. The conserved sequence falls within the previously defined P-region of voltage-activated K+ channels. In this study we investigate the effect of mutations in the signature sequence of the Shaker channel on K+ selectivity determined under bi-ionic conditions. Nonconservative substitutions of two threonine residues and the tyrosine residue leave selectivity intact. In contrast, mutations at some positions render the channel nonselective among monovalent cations. These findings are consistent with a proposal that the signature sequence contributes to a selectivity filter. Furthermore, the results illustrate that the hydroxyl groups at the third and fourth positions, and the aromatic group at position seven, are not essential in determining K+ selectivity.

Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1–S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5–S6, equivalent to M1–M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4–S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.

Potassium channels, a group of specialized membrane proteins, enable K+ ions to flow selectively across cell membranes. Transmembrane K+ currents underlie electrical signalling in neurons and other excitable cells. The atomic structure of a bacterial K+ channel pore has been solved by means of X-ray crystallography. To the extent that the prokaryotic pore is representative of other K+ channels, this landmark achievement has profound implications for our general understanding of K+ channels. But serious doubts have been raised concerning whether the prokaryotic K+ channel pore does actually represent those of eukaryotes. Here we have addressed this fundamental issue by substituting the prokaryotic pore into eukaryotic voltage-gated and inward-rectifier K+ channels. The resulting chimaeras retain the respective functional hallmarks of the eukaryotic channels, which indicates that the ion conduction pore is indeed conserved among K+ channels.

Inward-rectifier potassium channels conduct K+ across the cell membrane more efficiently in the inward than outward direction. This unusual conduction property is directly related to the biological action of these channels. One basis for inward rectification is voltage-dependent blockade by intracellular Mg2+ (refs 1, 7-9): strong inward-rectifier channels are so sensitive to intracellular Mg2+ that no outward K+ current is measurable under physiological conditions; weak inward rectifiers are less sensitive and allow some K+ to flow outwards. Background K1 channels and acetylcholine-regulated K+ channels from the heart are examples of strong inward rectifiers and ATP-sensitive K+ channels are weak rectifiers. Here we show that mutations at one position in the second transmembrane segment can alter the Mg2+ affinity and convert a weakly rectifying channel (ROMK1) into a strong rectifier. The amino acid at this position exposes its side chain to the aqueous pore and affects Mg2+ blockade as well as K+ conduction through an electrostatic mechanism.