Structural basis of gating modulation of Kv4 channel complexes

Modulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart1,2. Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary β-subunits—intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)—to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials1–5. However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2–DPP6S–KChIP1 dodecamer complex, the Kv4.2–KChIP1 and Kv4.2–DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2–KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1–S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2–KChIP1–DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex.

Auxiliary Subunits Regulate the Dendritic Turnover of AMPA Receptors in Mouse Hippocampal Neurons

Different families of auxiliary subunits regulate the function and trafficking of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the central nervous system. While a facilitatory role of auxiliary subunits in ER export and forward trafficking of newly synthesized AMPA receptors is firmly established, it is unclear whether auxiliary subunits also control endosomal receptor turnover in dendrites. Here, we manipulated the composition of AMPA receptor complexes in cultured hippocampal neurons by overexpression of two auxiliary subunits, transmembrane AMPAR regulatory protein (TARP) γ-8 or cysteine knot AMPAR-modulating protein (CKAMP) 44a, and monitored dendritic receptor cycling in live-cell imaging experiments. Receptor surface delivery was assayed using a modified AMPA receptor subunit carrying the pH-dependent fluorophore superecliptic pHluorin (SEP-GluA1), which regains its fluorescence during receptor exocytosis, when transiting from the acidic lumen of transport organelles to the neutral extracellular medium. Strikingly, we observed a dramatic reduction in the spontaneous fusion rate of AMPA receptor-containing organelles in neurons overexpressing either type of auxiliary subunit. An analysis of intracellular receptor distribution also revealed a decreased receptor pool in dendritic recycling endosomes, suggesting that incorporation of TARPγ-8 or CKAMP44a in receptor complexes generally diminishes cycling through the endosomal compartment. To directly analyze dendritic receptor turnover, we also generated a new reporter by N-terminal fusion of a self-labeling HaloTag to an AMPA receptor subunit (HaloTag-GluA1), which allows for selective, irreversible staining of surface receptors. Pulse chase-experiments with HaloTag-GluA1 indeed demonstrated that overexpression of TARPγ-8 or CKAMP44a reduces the constitutive internalization rate of surface receptors at extrasynaptic but not synaptic sites. Thus, our data point to a yet unrecognized regulatory function of TARPγ-8 and CKAMP44a, by which these structurally unrelated auxiliary subunits delay local recycling and increase surface lifetime of extrasynaptic AMPA receptors.

RNA polymerases from Low G+C Gram Positive Bacteria

The low G+C Gram positive bacteria represent some of the most medically and industrially important microorganisms. They are relied on for the production of food and dietary supplements, enzymes and antibiotics, as well as being responsible for the majority of nosocomial infections and serving as a reservoir for antibiotic resistance. Control of gene expression in this group is more highly studied than in any bacteria other than the Gram negative model Escherichia coli, yet until recently no structural information on RNA polymerase (RNAP) from this group was available. This review will summarise recent reports on the high resolution structure of RNAP from the model low G+C representative Bacillus subtilis, including the role of auxiliary subunits δ and ε, and outline approaches for the development of antimicrobials to target RNAP from this group.

Intrinsic Gating Behavior of Voltage-Gated Sodium Channels Predetermines Regulation by Auxiliary β-subunits

Voltage-gated sodium (Nav) channels mediate rapid millisecond electrical signaling in excitable cells. Auxiliary subunits, β1-β4, are thought to regulate Nav channel function through covalent and/or polar interactions with the channel’ s voltage-sensing domains. How these interactions translate into the diverse and variable regulatory effects of β-subunits remains unclear. Here, we find that the intrinsic movement order of the voltage-sensing domains during channel gating is unexpectedly variable across Nav channel isoforms. This movement order dictates the channel’ s propensity for closed-state inactivation, which in turn modulates the actions of β1 and β3. We show that the differential regulation of skeletal muscle, cardiac, and neuronal Nav channels is explained by their variable levels of closed-state inactivation. Together, this study provides a unified mechanism for the regulation of all Nav channel isoforms by β1 and β3, which explains how the fixed structural interactions of auxiliary subunits can paradoxically exert variable effects on channel function.