Ion channels

Abstract:

The superfamily of prokaryotic inwardly rectifying (KirBac) potassium channels is homologous to mammalian Kir channels. However, relatively little is known about their regulation or about their physiological role in vivo. In this study, we have used random mutagenesis and genetic complementation in K(+)-auxotrophic Escherichia coli and Saccharomyces cerevisiae to identify activatory mutations in a range of different KirBac channels. We also show that the KirBac6.1 gene (slr5078) is necessary for normal growth of the cyanobacterium Synechocystis PCC6803. Functional analysis and molecular dynamics simulations of selected activatory mutations identified regions within the slide helix, transmembrane helices, and C terminus that function as important regulators of KirBac channel activity, as well as a region close to the selectivity filter of KirBac3.1 that may have an effect on gating. In particular, the mutations identified in TM2 favor a model of KirBac channel gating in which opening of the pore at the helix-bundle crossing plays a far more important role than has recently been proposed.

Abstract:

Here we examined the gas-phase structures of two tetrameric membrane protein complexes by ion mobility mass spectrometry. The collision cross sections measured for the ion channel are in accord with a compact configuration of subunits, suggesting that the native-like structure can be preserved under the harsh activation conditions required to release it from the detergent micelle into the gas phase. We also found that the quaternary structure of the transporter, which has fewer transmembrane subunits than the ion channel, is less stable once stripped of detergents and bulk water. These results highlight the potential of ion mobility mass spectrometry for characterizing the overall topologies of membrane protein complexes and the structural changes associated with nucleotide, lipid, and drug binding.

Abstract:

Migraine with aura is a common, debilitating, recurrent headache disorder associated with transient and reversible focal neurological symptoms. A role has been suggested for the two-pore domain (K2P) potassium channel, TWIK-related spinal cord potassium channel (TRESK, encoded by KCNK18), in pain pathways and general anaesthesia. We therefore examined whether TRESK is involved in migraine by screening the KCNK18 gene in subjects diagnosed with migraine. Here we report a frameshift mutation, F139WfsX24, which segregates perfectly with typical migraine with aura in a large pedigree. We also identified prominent TRESK expression in migraine-salient areas such as the trigeminal ganglion. Functional characterization of this mutation demonstrates that it causes a complete loss of TRESK function and that the mutant subunit suppresses wild-type channel function through a dominant-negative effect, thus explaining the dominant penetrance of this allele. These results therefore support a role for TRESK in the pathogenesis of typical migraine with aura and further support the role of this channel as a potential therapeutic target.

Abstract:

Several inwardly-rectifying (Kir) potassium channels (Kin l 1, Kir41 and Kir4 2) are characterised by their sensitivity to inhibition by intracellular H+ within the physiological range The mechanism by which these channels are regulated by intracellular pH has been the subject of intense scrutiny for over a decade, yet the molecular identity of the titratable pH-sensor remains elusive In this study we have taken advantage of the acidic intracellular environment of S cerevisiae and used a K+-auxotrophic strain to screen for mutants of Kin 1 1 with impaired pH-sensitivity In addition to the previously identified K80M mutation, this unbiased screening approach identified a novel mutation (S172T) in the second transmembrane domain (TM2) that also produces a marked reduction in pH-sensitivity through destabilization of the closed-state However, despite this extensive mutagenic approach, no mutations could be identified which removed channel pH-sensitivity or which were likely to act as a separate H+-sensor unique to the pH-sensitive Kir channels In order to explain these results we propose a model in which the pH-sensing mechanism is part of an intrinsic gating mechanism common to all Kir channels not just the pH-sensitive Kin channels In this model mutations which disrupt this pH-sensor would result in an increase, not reduction, in pH-sensitivity This has major implications for any future studies of Kir channel pH-sensitivity and explains why formal identification of these pH-sensing residues still represents a major challenge