high-resolution-complexome-profiling-by-cryoslicing-bn-ms-analysis

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

University of Freiburg, Freiburg, Germany

Signalling Research Centres BIOSS and CIBSS, Freiburg, Germany

Large-conductance Ca(2+)- and voltage-activated potassium (BK(Ca)) channels shape the firing pattern in many types of excitable cell through their repolarizing K(+) conductance. The onset and duration of the BK(Ca)-mediated currents typically initiated by action potentials (APs) appear to be cell-type specific and were shown to vary between 1 ms and up to a few tens of milliseconds. In recent work, we showed that reliable activation of BK(Ca) channels under cellular conditions is enabled by their integration into complexes with voltage-activated Ca(2+) (Cav) channels that provide Ca(2+) ions at concentrations sufficiently high (> or =10 microM) for activation of BK(Ca) in the physiological voltage range. Formation of BK(Ca)-Cav complexes is restricted to a subset of Cav channels, Cav1.2 (L-type) and Cav2.1/2.2 (P/Q- and N-type), which differ greatly in their expression pattern and gating properties. Here, we reconstituted distinct BK(Ca)-Cav complexes in Xenopus oocytes and culture cells and used patch-clamp recordings to compare the functional properties of BK(Ca)-Cav1.2 and BK(Ca)-Cav2.1 complexes. Under steady-state conditions, K(+) currents mediated by BK(Ca)-Cav2.1 complexes exhibit a considerably faster rise time and reach maximum at potentials markedly more negative than complexes formed by BK(Ca) and Cav1.2, in line with the distinct steady-state activation and gating kinetics of the two Cav subtypes. When AP waveforms were used as a voltage command, K(+) currents mediated by BK(Ca)-Cav2.1 occurred at shorter APs and lasted longer than that of BK(Ca)-Cav1.2. These results demonstrate that the repolarizing K(+) currents through BK(Ca)-Cav complexes are shaped by the respective Cav subunit and that the distinct Cav channels may adapt BK(Ca) currents to the particular requirements of distinct types of cell.

Repolarizing responses of BKCa-Cav complexes are distinctly shaped by their Cav subunits.

Transient elevations in cytoplasmic Ca(2+) trigger a multitude of Ca(2+)-dependent processes in CNS neurons and many other cell types. The specificity, speed, and reliability of these processes is achieved and ensured by tightly restricting Ca(2+) signals to very local spatiotemporal domains, "Ca(2+) nano- and microdomains," that are centered around Ca(2+)-permeable channels. This arrangement requires that the Ca(2+)-dependent effectors reside within these spatial boundaries where the properties of the Ca(2+) domain and the Ca(2+) sensor of the effector determine the channel-effector activity. We use Ca(2+)-activated K(+) channels (K(Ca)) with either micromolar (BK(Ca) channels) or submicromolar (SK(Ca) channels) affinity for Ca(2+) ions to provide distance constraints for Ca(2+)-effector coupling in local Ca(2+) domains and review their significance for the cell physiology of K(Ca) channels in the CNS. The results may serve as a model for other processes operated by local Ca(2+) domains.

Control of K(Ca) channels by calcium nano/microdomains.

Molecular research on ion channels has demonstrated that many of these integral membrane proteins associate with partner proteins, often versatile in their function, or even assemble into stable macromolecular complexes that ensure specificity and proper rate of the channel-mediated signal transduction. Calcium-activated potassium (K(Ca)) channels that link excitability and intracellular calcium concentration are responsible for a wide variety of cellular processes ranging from regulation of smooth muscle tone to modulation of neurotransmission and control of neuronal firing pattern. Most of these functions are brought about by interaction of the channels' pore-forming subunits with distinct partner proteins. In this review we summarize recent insights into protein complexes associated with K(Ca) channels as revealed by proteomic research and discuss the results available on structure and function of these complexes and on the underlying protein-protein interactions. Finally, the results are related to their significance for the function of K(Ca) channels under cellular conditions.

Ca2+-activated K+ channels: from protein complexes to function.

Activation of K(+) channels by the G protein βγ subunits is an important signaling mechanism of G-protein-coupled receptors. Typically, receptor-activated K(+) currents desensitize in the sustained presence of agonists to avoid excessive effects on cellular activity. The auxiliary GABAB receptor subunit KCTD12 induces fast and pronounced desensitization of the K(+) current response. Using proteomic and electrophysiological approaches, we now show that KCTD12-induced desensitization results from a dual interaction with the G protein: constitutive binding stabilizes the heterotrimeric G protein at the receptor, whereas dynamic binding to the receptor-activated Gβγ subunits induces desensitization by uncoupling Gβγ from the effector K(+) channel. While receptor-free KCTD12 desensitizes K(+) currents activated by other GPCRs in vitro, native KCTD12 is exclusively associated with GABAB receptors. Accordingly, genetic ablation of KCTD12 specifically alters GABAB responses in the brain. Our results show that GABAB receptors are endowed with fast and reversible desensitization by harnessing KCTD12 that intercepts Gβγ signaling.

Auxiliary GABAB receptor subunits uncouple G protein βγ subunits from effector channels to induce desensitization.

Cornichon2 (CNIH2), an integral component of AMPA receptor (AMPAR) complexes in the mammalian brain, slows deactivation and desensitization of heterologously reconstituted receptor channels. Its significance in neuronal signal transduction, however, has remained elusive. Here we show by paired recordings that CNIH2-containing AMPARs dictate the slow decay of excitatory postsynaptic currents (EPSCs) elicited in hilar mossy cells of the hippocampus by single action potentials in mossy fiber boutons (MFB). Selective knockdown of CNIH2 markedly accelerated EPSCs in individual MFB-mossy cell synapses without altering the EPSC amplitude. In contrast, the rapidly decaying EPSCs in synapses between MFBs and aspiny interneurons that lack expression of CNIH2 were unaffected by the protein knockdown but were slowed by virus-directed expression of CNIH2. These results identify CNIH2 as the molecular distinction between slow and fast EPSC phenotypes and show that CNIH2 influences the time course and, hence, the efficacy of excitatory synaptic transmission.

Cornichon2 dictates the time course of excitatory transmission at individual hippocampal synapses.

Native AMPA receptors (AMPARs) in the mammalian brain are macromolecular complexes whose functional characteristics vary across the different brain regions and change during postnatal development or in response to neuronal activity. The structural and functional properties of the AMPARs are determined by their proteome, the ensemble of their protein building blocks. Here we use high-resolution quantitative mass spectrometry to analyze the entire pool of AMPARs affinity-isolated from distinct brain regions, selected sets of neurons, and whole brains at distinct stages of postnatal development. These analyses show that the AMPAR proteome is dynamic in both space and time: AMPARs exhibit profound region specificity in their architecture and the constituents building their core and periphery. Likewise, AMPARs exchange many of their building blocks during postnatal development. These results provide a unique resource and detailed contextual data sets for the analysis of native AMPAR complexes and their role in excitatory neurotransmission.

Regional diversity and developmental dynamics of the AMPA-receptor proteome in the mammalian brain.

Ionotropic AMPA-type glutamate receptors and G-protein-coupled metabotropic GABA receptors are key elements of neurotransmission whose cellular functions are determined by their protein constituents. Over the past couple of years unbiased proteomic approaches identified comprehensive sets of protein building blocks of these two types of neurotransmitter receptors in the brain (termed receptor proteomes). This provided the opportunity to match receptor proteomes with receptor physiology and to study the structural organization, regulation and function of native receptor complexes in an unprecedented manner. In this review we discuss the principles of receptor architecture and regulation emerging from the functional characterization of the proteomes of AMPA and GABA receptors. We also highlight progress in unraveling the role of unexpected protein components for receptor physiology.

Ionotropic AMPA-type glutamate and metabotropic GABA receptors: determining cellular physiology by proteomes.

Voltage-activated calcium (Cav) channels couple intracellular signaling pathways to membrane potential by providing Ca ions as second messengers at sufficiently high concentrations to modulate effector proteins located in the intimate vicinity of those channels. Here we show that protein kinase Cβ (PKCβ) and brain nitric oxide synthase (NOS1), both identified by proteomic analysis as constituents of the protein nano-environment of Cav2 channels in the brain, directly coassemble with Cav2.2 channels upon heterologous coexpression. Within Cav2.2-PKCβ and Cav2.2-NOS1 complexes voltage-triggered Ca influx through the Cav channels reliably initiates enzymatic activity within milliseconds. Using BK channels as target sensors for nitric oxide and protein phosphorylation together with high concentrations of Ca buffers showed that the complex-mediated Ca signaling occurs in local signaling domains at the plasma membrane. Our results establish Cav2-enzyme complexes as molecular entities for fast electrochemical coupling that reliably convert brief membrane depolarization into precisely timed intracellular signaling events in the mammalian brain.

Identification of Cav2-PKCβ and Cav2-NOS1 complexes as entities for ultrafast electrochemical coupling.

Plasma membrane Ca-ATPases (PMCAs), a family of P-type ATPases, extrude Ca ions from the cytosol to the extracellular space and are considered to be key regulators of Ca signaling. Here we show by functional proteomics that native PMCAs are heteromeric complexes that are assembled from two pore-forming PMCA1-4 subunits and two of the single-span membrane proteins, either neuroplastin or basigin. Contribution of the two Ig domain-containing proteins varies among different types of cells and along postnatal development. Complex formation of neuroplastin or basigin with PMCAs1-4 occurs in the endoplasmic reticulum and is obligatory for stability of the PMCA proteins and for delivery of PMCA complexes to the surface membrane. Knockout and (over)-expression of both neuroplastin and basigin profoundly affect the time course of PMCA-mediated Ca transport, as well as submembraneous Ca concentrations under steady-state conditions. Together, these results establish neuroplastin and basigin as obligatory auxiliary subunits of native PMCAs and key regulators of intracellular Ca concentration.

Article	Year
Repolarizing responses of BKCa-Cav complexes are distinctly shaped by their Cav subunits. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 18701686	2008
Control of K(Ca) channels by calcium nano/microdomains. Neuron\| PubMed ID: 18817728	2008
Ca2+-activated K+ channels: from protein complexes to function. Physiological reviews\| PubMed ID: 20959620	2010
Auxiliary GABAB receptor subunits uncouple G protein βγ subunits from effector channels to induce desensitization. Neuron\| PubMed ID: 24836506	2014
Cornichon2 dictates the time course of excitatory transmission at individual hippocampal synapses. Neuron\| PubMed ID: 24853943	2014
Regional diversity and developmental dynamics of the AMPA-receptor proteome in the mammalian brain. Neuron\| PubMed ID: 25242221	2014
Ionotropic AMPA-type glutamate and metabotropic GABA receptors: determining cellular physiology by proteomes. Current opinion in neurobiology\| PubMed ID: 28282540	2017
Identification of Cav2-PKCβ and Cav2-NOS1 complexes as entities for ultrafast electrochemical coupling. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 28507132	2017
Neuroplastin and Basigin Are Essential Auxiliary Subunits of Plasma Membrane Ca-ATPases and Key Regulators of Ca Clearance. Neuron\| PubMed ID: 29056295	2017

Bernd Fakler

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