[Show abstract][Hide abstract] ABSTRACT: Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4-S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4-S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules.
Full-text · Article · Mar 2015 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: KV10.1 is a voltage-gated potassium channel aberrantly expressed in many cases of cancer, and participates in cancer initiation
and tumor progression. Its action as an oncoprotein can be inhibited by a functional monoclonal antibody, indicating a role
for channels located at the plasma membrane, accessible to the antibody. Cortactin is an actin-interacting protein implicated
in cytoskeletal architecture and often amplified in several types of cancer. In this study, we describe a physical and functional
interaction between cortactin and KV10.1. Binding of these two proteins occurs between the C terminus of KV10.1 and the proline-rich domain of cortactin, regions targeted by many post-translational modifications. This interaction
is specific for KV10.1 and does not occur with KV10.2. Cortactin controls the abundance of KV10.1 at the plasma membrane and is required for functional expression of KV10.1 channels.
Preview · Article · Nov 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the
zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and
desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head
domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues,
six of these residues (N120–I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes
in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C
showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed
slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed
a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive
antagonist 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and
minor or no changes with the other mutants. Likewise, TNP-ATP–induced changes in control mutants distant from the proposed
ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed
ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We
also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.
Full-text · Article · Jul 2012 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: ATP-gated P2X receptors are trimeric ion channels that assemble as homo- or heteromers from seven cloned subunits. Transcripts and/or proteins of P2X subunits have been found in most, if not all, mammalian tissues and are being discovered in an increasing number of non-vertebrates. Both the first crystal structure of a P2X receptor and the generation of knockout (KO) mice for five of the seven cloned subtypes greatly advanced our understanding of their molecular and physiological function and their validation as drug targets. This review summarizes the current understanding of the structure and function of P2X receptors and gives an update on recent developments in the search for P2X subtype-selective ligands. It also provides an overview about the current knowledge of the regulation and modulation of P2X receptors on the cellular level and finally on their physiological roles as inferred from studies on KO mice.
Full-text · Article · May 2012 · Purinergic Signalling
[Show abstract][Hide abstract] ABSTRACT: K(V)10.1 is a mammalian brain voltage-gated potassium channel whose ectopic expression outside of the brain has been proven relevant for tumor biology. Promotion of cancer cell proliferation by K(V)10.1 depends largely on ion flow, but some oncogenic properties remain in the absence of ion permeation. Additionally, K(V)10.1 surface populations are small compared to large intracellular pools. Control of protein turnover within cells is key to both cellular plasticity and homeostasis, and therefore we set out to analyze how endocytic trafficking participates in controlling K(V)10.1 intracellular distribution and life cycle. To follow plasma membrane K(V)10.1 selectively, we generated a modified channel of displaying an extracellular affinity tag for surface labeling by α-bungarotoxin. This modification only minimally affected K(V)10.1 electrophysiological properties. Using a combination of microscopy and biochemistry techniques, we show that K(V)10.1 is constitutively internalized involving at least two distinct pathways of endocytosis and mainly sorted to lysosomes. This occurs at a relatively fast rate. Simultaneously, recycling seems to contribute to maintain basal K(V)10.1 surface levels. Brief K(V)10.1 surface half-life and rapid lysosomal targeting is a relevant factor to be taken into account for potential drug delivery and targeting strategies directed against K(V)10.1 on tumor cells.
[Show abstract][Hide abstract] ABSTRACT: The ether-à-go-go potassium channels hEag1 and hEag2 are highly homologous. Even though both possess identical voltage-sensing domain S4, the channels act differently in response to voltage. Therefore we asked whether transmembrane domains other than the voltage sensor could contribute to the voltage-dependent behaviour of these potassium channels. For this chimaeras were created, in which each single transmembrane domain of hEag1 was replaced by the corresponding segment of hEag2. The voltage-dependent properties of the chimaeras were analysed after expression in Xenopus laevis oocytes using the two-electrode voltage-clamp method. By this we found, that only the mutations in transmembrane domains S5 and S6 are able to change the voltage sensitivity of hEag1 by shifting the half-activation potential (V
50) to values intermediate between the two wild types. Moreover, the presence of Mg2+ has strong effects on the voltage sensitivity of hEag2 shifting V
50 by more than 50 mV to more positive values. Interestingly, despite the identical binding site Mg2+ showed only little effects on hEag1 or the chimaeras. Altogether, our data suggest that not only transmembrane spanning regions, but also non-membrane spanning regions are responsible for differences in the behaviour of the hEag1 and hEag2 potassium channels.
Full-text · Article · May 2008 · Biophysics of Structure and Mechanism