The M1P1 loop of TASK3 K2P channels apposes the selectivity filter and influences channel function.

Channels of the two-pore domain potassium (K2P) family contain two pore domains rather than one and an unusually long pre-pore extracellular linker called the M1P1 loop. The TASK (TASK1, TASK3, and TASK5) subfamily of K2P channels is regulated by a number of different pharmacological and physiological mediators. At pH 7.4 TASK3 channels are selectively blocked by zinc in a manner that is both pH(o)- and [K](o)(-)dependent. Mutation of both the Glu-70 residue in the M1P1 loop and the His-98 residue in the pore region abolished block, suggesting the two residues may contribute to a zinc binding site. Mutation of one Glu-70 residue and one His-98 residue to cysteine in TASK3 fixed concatamer channels gave currents that were enhanced by dithiothreitol and then potently blocked by cadmium, suggesting that spontaneous disulfide bridges could be formed between these two residues. Swapping the M1P1 loops of TASK1 and TASK3 channels showed that the M1P1 loop is also involved in channel regulation by pH. Therefore, the TASK3 M1P1 loop lies close to the pore, regulating TASK3 channel activity.

Molecular basis of slow activation of the human ether-a-go-go related gene potassium channel.

The human ether-á-go-go related gene (HERG) encodes the pore forming alpha-subunit of the rapid delayed rectifier K(+) channel which is central to the repolarization phase of the cardiac action potential. HERG K(+) channels have unusual kinetics characterized by slow activation and deactivation, yet rapid inactivation. The fourth transmembrane domain (S4) of HERG, like other voltage-gated K(+) channels, contains multiple positive charges and is the voltage sensor for activation. In this study, we mutated each of the positively charged residues in this region to glutamine (Q), expressed the mutant and wild-type (WT) channels in Xenopus laevis oocytes and studied them using two-electrode voltage clamp methods. K525Q channels activated at more hyperpolarized potentials than WT, whereas all the other mutant channels activated at more depolarized potentials. All mutants except for R531Q also had a reduction in apparent gating charge associated with activation. Mutation of K525 to cysteine (C) resulted in a less dramatic phenotype than K525Q. The addition of the positively charged MTSET to K525C altered the phenotype to one more similar to K525Q than to WT. Therefore it is not charge per se, but the specific lysine side chain at position 525, that is crucial for stabilizing the closed state. When rates of activation and deactivation for WT and mutant channels were compared at equivalent total (chemical + electrostatic) driving forces, K525Q and R528Q accelerated activation but had no effect on deactivation, R531Q slowed activation and deactivation, R534Q accelerated activation but slowed deactivation and R537Q accelerated deactivation but had no effect on activation. The main conclusions we can draw from these data are that in WT channels K525 stabilizes the closed state, R531 stabilizes the open state and R534 participates in interactions that stabilize pre-open closed states.

Structure of the HERG K+ channel S5P extracellular linker: role of an amphipathic alpha-helix in C-type inactivation.

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp585-Ile593 and Gly604-Tyr611 of the channel. The Trp585-Ile593 helix has distinct hydrophilic and hydrophobic surfaces. The Gly604-Tyr611 helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.