The Brain-Specific Beta4 Subunit Downregulates BK Channel Cell Surface Expression

The large-conductance K+ channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca++- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking

Somatostatin-expressing interneurons modulate neocortical network through GABAb receptors in a synapse-specific manner

Abstract The firing activity of somatostatin-expressing inhibitory neurons (SST-INs) can suppress network activity via both GABAa and GABAb receptors (Rs). Although SST-INs do not receive GABAaR input from other SST-INs, it is possible that SST-IN-released GABA could suppress the activity of SST-INs themselves via GABAbRs, providing a negative feedback loop. Here we characterized the influence of GABAbR modulation on SST-IN activity in layer 2/3 of the somatosensory cortex in mice. We compared this to the effects of GABAbR activation on parvalbumin-expressing interneurons (PV-INs). Using in vitro whole-cell patch clamp recordings, pharmacological and optogenetic manipulations, we found that the firing activity of SST-INs suppresses excitatory drive to themselves via presynaptic GABAbRs. Postsynaptic GABAbRs did not influence SST-IN spontaneous activity or intrinsic excitability. Although GABAbRs at pre- and postsynaptic inputs to PV-INs are modestly activated during cortical network activity in vitro, the spontaneous firing of SST-INs was not the source of GABA driving this GABAbR activation. Thus, SST-IN firing regulates excitatory synaptic strength through presynaptic GABAbRs at connections between pyramidal neurons (Pyr-Pyr) and synapses between pyramidal neurons and SST-INs (Pyr-SST), but not Pyr-PV and PV-Pyr synapses. Our study indicates that two main types of neocortical inhibitory interneurons are differentially modulated by SST-IN-mediated GABA release

Presentation_1_GABAAR-mediated tonic inhibition differentially modulates intrinsic excitability of VIP- and SST- expressing interneurons in layers 2/3 of the somatosensory cortex.PPTX

Extrasynaptic GABAA receptors (GABAARs) mediating tonic inhibition are thought to play an important role in the regulation of neuronal excitability. However, little is known about a cell type-specific tonic inhibition in molecularly distinctive types of GABAergic interneurons in the mammalian neocortex. Here, we used whole-cell patch-clamp techniques in brain slices prepared from transgenic mice expressing red fluorescent protein (TdTomato) in vasoactive intestinal polypeptide- or somatostatin- positive interneurons (VIP-INs and SST-INs, respectively) to investigate tonic and phasic GABAAR-mediated inhibition as well as effects of GABAA inhibition on intrinsic excitability of these interneurons in layers 2/3 (L2/3) of the somatosensory (barrel) cortex. We found that tonic inhibition was stronger in VIP-INs compared to SST-INs. Contrary to the literature data, tonic inhibition in SST-INs was comparable to pyramidal (Pyr) neurons. Next, tonic inhibition in both interneuron types was dependent on the activity of delta subunit-containing GABAARs. Finally, the GABAAR activity decreased intrinsic excitability of VIP-INs but not SST-INs. Altogether, our data indicate that GABAAR-mediated inhibition modulates neocortical interneurons in a type-specific manner. In contrast to L2/3 VIP-INs, intrinsic excitability of L2/3 SST-INs is immune to the GABAAR-mediated inhibition.</p

Differential wiring of layer 2/3 neurons drives sparse and reliable firing during neocortical development.

<p>Sensory information is transmitted with high fidelity across multiple synapses until it reaches the neocortex. There, individual neurons exhibit enormous variability in responses. The source of this diversity in output has been debated. Using transgenic mice expressing the green fluorescent protein coupled to the activity-dependent gene c-fos, we identified neurons with a history of elevated activity in vivo. Focusing on layer 4 to layer 2/3 connections, a site of strong excitatory drive at an initial stage of cortical processing, we find that fluorescently tagged neurons receive significantly greater excitatory and reduced inhibitory input compared with neighboring, unlabeled cells. Differential wiring of layer 2/3 neurons arises early in development and requires sensory input to be established. Stronger connection strength is not associated with evidence for recent synaptic plasticity, suggesting that these more active ensembles may not be generated over short time scales. Paired recordings show fosGFP+ neurons spike at lower stimulus thresholds than neighboring, fosGFP- neurons. These data indicate that differences in circuit construction can underlie response heterogeneity amongst neocortical neurons.</p

The brain-specific Beta4 subunit downregulates BK channel cell surface expression.

<p>The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.</p

Genetic ablation of β4 results in larger BK channel currents in CA3 pyramidal neurons.

<p>(A) Fluorescent image of a living slice from a β4−/− knock-out mouse showing GFP-signal in hippocampus. Scale bar: 500 µm. (B) Mean total potassium current from β4 heterozygote (β4+/−) and β4 knock-out (β4−/−) mice. (C) Example paxilline-sensitive BK channel current from representative CA3 neurons in β4+/− and β4−/− mice. (D) Mean amplitude of paxilline-sensitive BK channel current in β4+/− and β4−/− mice.</p

An ER-retention/retrieval sequence at the C-terminus of β4 inhibits the surface expression of BKα and β4.

<p>(A) Schematic of the mutant β4 constructs showing the C-terminal amino acids. (B) Fluorescence from binding of cell-impermeable dye to cell-surface BKα channels in cells transfected with FAP-BKα (red) and GFP (green). (C) Same as (B) but in cells transfected with FAP-BKα and wild-type β4. (D) Same as (B) but in cells transfected with FAP-BKα and β4-Ala. (E) Same as (B) but in cells transfected with FAP-BKα and β4-polySer. Scale bar = 20 µm (B–E). (F) Mean intensity of surface fluorescence for β4 constructs normalized to fluorescence from cells transfected with FAP-BKα alone. (G) Histogram showing distribution of fluorescence intensity in cells transfected with FAP-BKα alone (red), FAP-BKα+β4-Ala (green) and FAP-BKα+β4-polySer (blue). Scale bar: 20 µm (B–E).</p

Co-expression of the β4 subunit reduces cell-surface trafficking of BK channels.

<p>(A) Membrane topology of the FAP-BKα and β4 proteins. The FAP tag is at the extracellular, N-terminus. The C-terminus of the BKα subunit is indicated. (B) Schematic of binding of cell-impermeable dye (pink) to the FAP results in significant increase in fluorescence (red). (C) Application of cell-impermeable dye labels only surface BKα channels in live HEK-293 cells co-transfected with FAP-BKα (red) and GFP (green). (D) Same as (C) but in cells co-transfected with β4, FAP-BKα, and GFP showing reduced surface expressed of the FAP-BKα. (E) Application of cell-permeable dye labels intracellular stores of channel in cells transfected with FAP-BKα (red) and GFP (green). (F) Same as (E) but in cells transfected with β4, FAP-BKα, and GFP. Scale bar = 20 µm (C–F). (G) Distribution of surface fluorescence intensity values after application of cell-impermeable dye in transfected cells for FAP-BKα (red bars) or FAP-BKα+β4 (black bars). (H–I) Proposed model for surface distribution of BK channels in the absence (H) or presence (I) of the β4 subunit.</p