The N–Terminal Tail of hERG Contains an Amphipathic α–Helix That Regulates Channel Deactivation

The cytoplasmic N–terminal domain of the human ether–a–go–go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N–terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N–terminal 135 residues of hERG contains a previously described Per–Arnt–Sim (PAS) domain (residues 26–135) as well as an amphipathic α–helix (residues 13–23) and an initial unstructured segment (residues 2–9). Deletion of residues 2–25, only the unstructured segment (residues 2–9) or replacement of the α–helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α–helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N–terminal α–helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel

The Bicarbonate Transporter Is Essential for Bacillus anthracis Lethality

In the pathogenic bacterium Bacillus anthracis, virulence requires induced expression of the anthrax toxin and capsule genes. Elevated CO2/bicarbonate levels, an indicator of the host environment, provide a signal ex vivo to increase expression of virulence factors, but the mechanism underlying induction and its relevance in vivo are unknown. We identified a previously uncharacterized ABC transporter (BAS2714-12) similar to bicarbonate transporters in photosynthetic cyanobacteria, which is essential to the bicarbonate induction of virulence gene expression. Deletion of the genes for the transporter abolished induction of toxin gene expression and strongly decreased the rate of bicarbonate uptake ex vivo, demonstrating that the BAS2714-12 locus encodes a bicarbonate ABC transporter. The bicarbonate transporter deletion strain was avirulent in the A/J mouse model of infection. Carbonic anhydrase inhibitors, which prevent the interconversion of CO2 and bicarbonate, significantly affected toxin expression only in the absence of bicarbonate or the bicarbonate transporter, suggesting that carbonic anhydrase activity is not essential to virulence factor induction and that bicarbonate, and not CO2, is the signal essential for virulence induction. The identification of this novel bicarbonate transporter essential to virulence of B. anthracis may be of relevance to other pathogens, such as Streptococcus pyogenes, Escherichia coli, Borrelia burgdorferi, and Vibrio cholera that regulate virulence factor expression in response to CO2/bicarbonate, and suggests it may be a target for antibacterial intervention

The S4–S5 Linker Acts as a Signal Integrator for hERG K+ Channel Activation and Deactivation Gating

Human ether-à-go-go-related gene (hERG) K+ channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4–S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4–S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4–S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4–S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4–S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4–S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel

Gasometric Analysis in Plant Investigation

RESP-3540