Eitan Reuveny

tBID Homooligomerizes in the Mitochondrial Membrane to Induce Apoptosis

Activation of the tumor necrosis factor R1/Fas receptor results in the cleavage of cytosolic BID to truncated tBID. tBID translocates to the mitochondria to induce the oligomerization of BAX or BAK, resulting in the release of cytochromec (Cyt c). Here we demonstrate that in tumor necrosis factor α-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex that does not include BAX or BAK. Using fluorescence resonance energy transfer analysis and co-immunoprecipitation, we demonstrate that tBID-tBID interactions occur in the mitochondria of living cells. Cross-linking experiments using a tBID-GST chimera indicated that tBID forms homotrimers in the mitochondrial membrane. To test the functional consequence of tBID oligomerization, we expressed a chimeric FKBP-tBID molecule. Enforced dimerization of FKBP-tBID by the bivalent ligand FK1012 resulted in Cytc release, caspase activation, and apoptosis. Surprisingly, enforced dimerization of tBID did not result in the dimerization of either BAX or BAK. Moreover, a tBID BH3 mutant (G94E), which does not interact with or induce the dimerization of either BAX or BAK, formed the 45-kDa complex and induced both Cyt c release and apoptosis. Thus, tBID oligomerization may represent an alternative mechanism for inducing mitochondrial dysfunction and apoptosis.

Conformational Rearrangements Associated with the Gating of the G Protein-Coupled Potassium Channel Revealed by FRET Microscopy

G protein-coupled potassium channels (GIRK/Kir3.x) are key determinants that translate inhibitory chemical neurotransmission into changes in cellular excitability. To understand the mechanism of channel activation by G proteins, it is necessary to define the structural rearrangements in the channel that result from interaction with Gbetagamma subunits. In this study we used a combination of fluorescence spectroscopy and through-the-objective total internal reflection microscopy to monitor the conformational rearrangements associated with the activation of GIRK channels in single intact cells. We detect activation-induced changes in FRET consistent with a rotation and expansion of the termini along the central axis of the channel. We propose that this rotation and expansion of the termini drives the channel to open by bending and possibly rotating the second transmembrane segment.

GIRK Channel Activation Involves a Local Rearrangement of a Preformed G Protein Channel Complex

G protein-coupled signaling is one of the major mechanisms for controlling cellular excitability. One of the main targets for this control at postsynaptic membranes is the G protein-coupled potassium channels (GIRK/Kir3), which generate slow inhibitory postsynaptic potentials following the activation of Pertussis toxin-sensitive G protein-coupled receptors. Using total internal reflection fluorescence (TIRF) microscopy combined with fluorescence resonance energy transfer (FRET), in intact cells, we provide evidence for the existence of a trimeric G protein-channel complex at rest. We show that activation of the channel via the receptor induces a local conformational switch of the G protein to induce channel opening. The presence of such a complex thus provides the means for a precise temporal and highly selective activation of the channel, which is required for fine tuning of neuronal excitability.

Gating of GIRK Channels: Details of an Intricate, Membrane-Delimited Signaling Complex

G protein-coupled inwardly rectifying potassium channels (GIRK/Kir3) are important elements in controlling cellular excitability. In recent years, tremendous progress has been made toward understanding various components involved in channel activation, modulation, and signaling specificity. In this review, we summarize these recent findings and attempt to put them in context with recently available structural data.

Coupling Gβγ-Dependent Activation to Channel Opening via Pore Elements in Inwardly Rectifying Potassium Channels

Abstract G protein–coupled inwardly rectifying potassium channels, GIRK/Kir3.x, are gated by the Gβγ subunits of the G protein. The molecular mechanism of gating was investigated by employing a novel yeast-based random mutagenesis approach that selected for channel mutants that are active in the absence of Gβγ. Mutations in TM2 were found that mimicked the Gβγ-activated state. The activity of these channel mutants was independent of receptor stimulation and of the availability of heterologously expressed Gβγ subunits but depended on PtdIns(4,5)P 2 . The results suggest that the TM2 region plays a key role in channel gating following Gβγ binding in a phospholipid-dependent manner. This mechanism of gating in inwardly rectifying K + channels may be similar to the involvement of the homologous region in prokaryotic KcsA potassium channel and, thus, suggests evolutionary conservation of the gating structure.

MinireviewGating of GIRK Channels: Details of an Intricate, Membrane-Delimited Signaling Complex

G protein-coupled inwardly rectifying potassium channels (GIRK/Kir3) are important elements in controlling cellular excitability. In recent years, tremendous progress has been made toward understanding various components involved in channel activation, modulation, and signaling specificity. In this review, we summarize these recent findings and attempt to put them in context with recently available structural data.

Mechanism of Ba2+ block of a mouse inwardly rectifying K+ channel: differential contribution by two discrete residues

1 The block of the IRK1/Kir2.1 inwardly rectifying K+ channel by a Ba2+ ion is highly voltage dependent, where the ion binds approximately half‐way within the membrane electrical field. The mechanism by which two distinct mutations, E125N and T141A, affect Ba2+ block of Kir2.1 was investigated using heterologous expression in Xenopus oocytes. 2 Analysis of the blocking kinetics showed that E125 and T141 affect the entry and binding of Ba2+ to the channel, respectively. Replacing the glutamate at position 125 with an asparagine greatly decreased the rate at which the Ba2+ ions enter and leave the pore. In contrast, replacing the polar threonine at position 141 with an alanine affected the entry rate of the Ba2+ ions while leaving the exit rate unchanged. 3 Acidification of the extracellular solution slowed the exit rate of the Ba2+ from the wild‐type channel, but had no such effect on the Kir2.1(E125N) mutant. 4 These results thus reveal two unique roles for the amino acids at positions 125 and 141 in aiding the interaction of Ba2+ with the channel. Their possible roles in K+ permeation are discussed.

Fast inactivation of a brain K+ channel composed of Kv1.1 and Kvβ1.1 subunits modulated by G protein βγ subunits

Modulation of A‐type voltage‐gated K+ channels can produce plastic changes in neuronal signaling. It was shown that the delayed‐rectifier Kv1.1 channel can be converted to A‐type upon association with Kvβ1.1 subunits; the conversion is only partial and is modulated by phosphorylation and microfilaments. Here we show that, in Xenopus oocytes, expression of Gβ1γ2 subunits concomitantly with the channel (composed of Kv1.1 and Kvβ1.1 subunits), but not after the channels expression in the plasma membrane, increases the extent of conversion to A‐type. Conversely, scavenging endogenous Gβγ by co‐expression of the C‐terminal fragment of the β‐adrenergic receptor kinase reduces the extent of conversion to A‐type. The effect of Gβγ co‐expression is occluded by treatment with dihydrocytochalasin B, a microfilament‐disrupting agent shown previously by us to enhance the extent of conversion to A‐type, and by overexpression of Kvβ1.1. Gβ1γ2 subunits interact directly with GST fusion fragments of Kv1.1 and Kvβ1.1. Co‐expression of Gβ1γ2 causes co‐immunoprecipitation with Kv1.1 of more Kvβ1.1 subunits. Thus, we suggest that Gβ1γ2 directly affects the interaction between Kv1.1 and Kvβ1.1 during channel assembly which, in turn, disrupts the ability of the channel to interact with microfilaments, resulting in an increased extent of A‐type conversion.

Modulation of Basal and Receptor-Induced GIRK Potassium Channel Activity and Neuronal Excitability by the Mammalian PINS Homolog LGN

G protein-activated inwardly rectifying potassium (GIRK) channels mediate slow synaptic inhibition and control neuronal excitability. It is unknown whether GIRK channels are subject to regulation by guanine dissociation inhibitor (GDI) proteins like LGN, a mammalian homolog of Drosophila Partner of Inscuteable (mPINS). Here we report that LGN increases basal GIRK current but reduces GIRK activation by metabotropic transmitter receptors coupled to Gi or Go, but not Gs. Moreover, expression of its N-terminal, TPR-containing protein interaction domains mimics the effects of LGN in mammalian cells, probably by releasing sequestered endogenous LGN. In hippocampal neurons, expression of LGN, or LGN fragments that mimic or enhance LGN activity, hyperpolarizes the resting potential due to increased basal GIRK activity and reduces excitability. Using Lenti virus for LGN RNAi to reduce endogenous LGN levels in hippocampal neurons, we further show an essential role of LGN for maintaining basal GIRK channel activity and for harnessing neuronal excitability.

The Pore Helix Dipole Has a Minor Role in Inward Rectifier Channel Function

Ion channels lower the energetic barrier for ion passage across cell membranes and enable the generation of bioelectricity. Electrostatic interactions between permeant ions and channel pore helix dipoles have been proposed as a general mechanism for facilitating ion passage. Here, using genetic selections to probe interactions of an exemplar potassium channel blocker, barium, with the inward rectifier Kir2.1, we identify mutants bearing positively charged residues in the potassium channel signature sequence at the pore helix C terminus. We show that these channels are functional, selective, resistant to barium block, and have minimally altered conductance properties. Both the experimental data and model calculations indicate that barium resistance originates from electrostatics. We demonstrate that potassium channel function is remarkably unperturbed when positive charges occur near the permeant ions at a location that should counteract pore helix electrostatic effects. Thus, contrary to accepted models, the pore helix dipole seems to be a minor factor in potassium channel permeation.