Risk stratification of EGFR+ lung cancer diagnosed with panel-based next-generation sequencing.

OBJECTIVE: Panel-based next-generation sequencing (NGS) is increasingly used for the diagnosis of EGFR-mutated non-small-cell lung cancer (NSCLC) and could improve risk assessment in combination with clinical parameters. MATERIALS AND METHODS: To this end, we retrospectively analyzed the outcome of 400 tyrosine kinase inhibitor (TKI)-treated EGFR+ NSCLC patients with validation of results in an independent cohort (n = 130). RESULTS: EGFR alterations other than exon 19 deletions (non-del19), TP53 co-mutations, and brain metastases at baseline showed independent associations of similar strengths with progression-free (PFS hazard ratios [HR] 2.1-2.3) and overall survival (OS HR 1.7-2.2), in combination defining patient subgroups with distinct outcome (EGFR+NSCLC risk Score, "ENS", p < 0.001). Co-mutations beyond TP53 were rarely detected by our multigene panel (<5%) and not associated with clinical endpoints. Smoking did not affect outcome independently, but was associated with non-del19 EGFR mutations (p < 0.05) and comorbidities (p < 0.001). Laboratory parameters, like the blood lymphocyte-to-neutrophil ratio and serum LDH, correlated with the metastatic pattern (p < 0.01), but had no independent prognostic value. Reduced ECOG performance status (PS) was associated with comorbidities (p < 0.05) and shorter OS (p < 0.05), but preserved TKI efficacy. Non-adenocarcinoma histology was also associated with shorter OS (p < 0.05), but rare (2-3 %). The ECOG PS and non-adenocarcinoma histology could not be validated in our independent cohort, and did not increase the range of prognostication alongside the ENS. CONCLUSIONS: EGFR variant, TP53 status and brain metastases predict TKI efficacy and survival in EGFR+ NSCLC irrespective of other currently available parameters ("ENS"). Together, they constitute a practical and reproducible approach for risk stratification of newly diagnosed metastatic EGFR+ NSCLC.

Generating bursts (and pauses) in the dopamine midbrain neurons.

Dopamine (DA) midbrain neurons project to several striatal and cortical target areas and are essentially involved in a puzzling variety of important brain functions such as action selection and motor performance, motivation and reward-based learning, but also working memory and cognition. These neurons act via the release of their (main) neurotransmitter, dopamine, which binds to metabotropic dopamine receptors of the D1 or D2 type on target neurons. Axonal but also dendritic dopamine release is essentially controlled by calcium-triggered exocytosis of dopamine-filled synaptic vesicles primarily driven by electrical activity of the dopamine neuron, which generates patterns of actions potentials in the somato-dendritic domain and distributes them along its axonal tree. Thus, recording the behaviorally relevant pattern of electrical activity in DA neurons and identifying the underlying biophysical mechanisms that integrate afferent synaptic inputs and intrinsic excitability constitute a crucial element for defining the physiological roles of the midbrain DA system. Electrical activity of midbrain DA neurons in vivo is characterized by tonic background activity in a narrow frequency range (ca. 1-8Hz) interrupted by either transient (i.e. phasic, <500ms) sequences of high-frequency firing (>15Hz), so called "bursts", or transient pauses of electrical activity, where DA neurons generate no action potentials. This review focuses on the properties of these phasic activity changes in midbrain DA neurons. It updates recent progress on the expanding behavioral contexts, associated with phasic electrical activity in DA neurons beyond the classical (canonical) reward prediction error model. The review also highlights recently defined contributions of synaptic inputs for burst and pause generation and the roles of distinct postsynaptic ion channels in midbrain DA neurons.

Tuning pacemaker frequency of individual dopaminergic neurons by Kv4.3L and KChip3.1 transcription.

The activity of dopaminergic (DA) substantia nigra (SN) neurons is essential for voluntary movement control. An intrinsic pacemaker in DA SN neurons generates their tonic spontaneous activity, which triggers dopamine release. We show here, by combining multiplex and quantitative real-time single-cell RT- PCR with slice patch-clamp electrophysiology, that an A-type potassium channel mediated by Kv4.3 and KChip3 subunits has a key role in pacemaker control. The number of active A-type potassium channels is not only tightly associated with the pacemaker frequency of individual DA SN neurons, but is also highly correlated with their number of Kv4.3L (long splice variant) and KChip3.1 (long splice variant) mRNA molecules. Consequently, the variation of Kv4alpha and Kv4beta subunit transcript numbers is sufficient to explain the full spectrum of spontaneous pacemaker frequencies in identified DA SN neurons. This linear coupling between Kv4alpha as well as Kv4beta mRNA abundance, A-type channel density and pacemaker frequency suggests a surprisingly simple molecular mechanism for how DA SN neurons tune their variable firing rates by transcriptional control of ion channel genes.

ATP-sensitive potassium channels in dopaminergic neurons: transducers of mitochondrial dysfunction.

ATP-sensitive potassium (K(ATP)) channels directly couple the metabolic state of a cell to its electrical activity. Dopaminergic midbrain neurons express alternative types of K(ATP) channels mediating their differential response to mitochondrial complex I inhibition. Because reduced complex I activity is present in Parkinson's Disease, differential K(ATP) channel expression suggests a novel candidate mechanism for selective dopaminergic degeneration.

Molecular physiology of neuronal K-ATP channels (review).

ATP sensitive potassium (K-ATP) channels are widely expressed in many cell types including neurons. K-ATP channels are heteromeric membrane proteins that consist of two very different subunits: the pore-forming, two-transmembrane spanning potassium channel subunit (Kir6) and the regulatory, 17 transmembrane spanning sulphonylurea receptor (SUR). This ensemble--joined together in a 4:4 stoichiometry--endows this channel with a unique combination of functional properties. The open probability of K-ATP channels directly depends on the intracellular ATP/ADP levels allowing the channels to directly couple the metabolic state of a cell to its electrical activity. Here, recent progress on the molecular composition and functional diversity of neuronal K-ATP channels is reviewed. One is particular concerned with single-cell mRNA expression studies that give insight to the coexpression patterns of Kir6 and SUR isoforms in identified neurons. In addition, the physiological roles of neuronal K-ATP channels in glucose sensing and adapting neuronal activity to metabolic demands are discussed, as well as their emerging pathophysiological functions in acute brain ischemia and chronic neurodegenerative diseases.

Differential expression of the small-conductance, calcium-activated potassium channel SK3 is critical for pacemaker control in dopaminergic midbrain neurons.

The physiological activity of dopaminergic midbrain (DA) neurons is important for movement, cognition, and reward. Altered activity of DA neurons is a key finding in schizophrenia, but the cellular mechanisms have not been identified. Recently, KCNN3, a gene that encodes a member (SK3) of the small-conductance, calcium-activated potassium (SK) channels, has been proposed as a candidate gene for schizophrenia. However, the functional role of SK3 channels in DA neurons is unclear. We combined patch-clamp recordings with single-cell RT-PCR and confocal immunohistochemistry in mouse midbrain slices to study the function of molecularly defined SK channels in DA neurons. Biophysical and pharmacological analysis, single-cell mRNA, and protein expression profiling strongly suggest that SK3 channels mediate the calcium-dependent afterhyperpolarization in DA neurons. Perforated patch recordings of DA neurons in the substantia nigra (SN) demonstrated that SK3 channels dynamically control the frequency of spontaneous firing. In addition, SK3 channel activity was essential to maintain the high precision of the intrinsic pacemaker of DA SN neurons. In contrast, in the ventral tegmental area, DA neurons displayed significantly smaller SK currents and lower SK3 protein expression. In these DA neurons, SK3 channels were not involved in pacemaker control. Accordingly, they discharged in a more irregular manner compared with DA SN neurons. Thus, our study shows that differential SK3 channel expression is a critical molecular mechanism in DA neurons to control neuronal activity. This provides a cellular framework to understand the functional consequences of altered SK3 expression, a candidate disease mechanism for schizophrenia.

ATP-sensitive K+ channels in the hypothalamus are essential for the maintenance of glucose homeostasis.

Glucose-responsive (GR) neurons in the hypothalamus are thought to be critical in glucose homeostasis, but it is not known how they function in this context. Kir6.2 is the pore-forming subunit of K(ATP) channels in many cell types, including pancreatic beta-cells and heart. Here we show the complete absence of both functional ATP-sensitive K+ (K(ATP)) channels and glucose responsiveness in the neurons of the ventromedial hypothalamus (VMH) in Kir6.2-/- mice. Although pancreatic alpha-cells were functional in Kir6.2-/-, the mice exhibited a severe defect in glucagon secretion in response to systemic hypoglycemia. In addition, they showed a complete loss of glucagon secretion, together with reduced food intake in response to neuroglycopenia. Thus, our results demonstrate that KATP channels are important in glucose sensing in VMH GR neurons, and are essential for the maintenance of glucose homeostasis.

Single-cell mRNA expression of HCN1 correlates with a fast gating phenotype of hyperpolarization-activated cyclic nucleotide-gated ion channels (Ih) in central neurons.

Hyperpolarization-activated currents (Ih) are key players in shaping rhythmic neuronal activity. Although candidate genes for Ih channels have been cloned (HCN1-HCN4), the subunit composition of different native Ih channels is unknown. We used a combined patch-clamp and qualitative single-cell reverse transcription multiplex polymerase chain reaction (RT-mPCR) approach to analyse HCN1-4 coexpression profiles in four neuronal populations in mouse CNS. Coexpression of HCN2, HCN3 and HCN4 mRNA was detected in single neurons of all four neuronal cell types analysed. In contrast, HCN1 mRNA was detected in neocortical and hippocampal pyramidal neurons but not in dopaminergic midbrain and thalamocortical neurons. HCN1 expression was correlated with significantly faster activation kinetics on the level of individual neurons. Semiquantitative single-cell RT-mPCR analysis demonstrated that HCN1 mRNA expression is at least eightfold higher in cortical neurons than subcortical neurons. We show that single neurons possess complex coexpression patterns of Ih candidate genes. Alternative expression of HCN1 is likely to be an important molecular determinant to generate the different neuronal Ih channel species adapted to tune either subcortical or cortical network activity.

Activity-dependent formation of perforated synapses in cultured hippocampal neurons.

The study investigated the formation of perforated synapses in rat hippocampal cell cultures. Perforated synapses are defined by their discontinuous postsynaptic densities (PSDs) and are believed to occur in parallel with changes in synaptic activity and possibly also synaptic efficacy. Several in vivo studies have demonstrated an increase in the frequency of perforated synapses induced by development and environmental stimulation as well as long-term potentiation (LTP). Also in in vitro brain slices, LTP was associated with an elevated number of perforated spine synapses. Our study demonstrated for the first time that the formation of perforated synapses can be induced by a short-term increase in spontaneous neural activity in a hippocampal cell culture model. Stimulation with the GABAA-antagonist picrotoxin (PTX) induced a significant increase in the percentage of perforated synapses. This strong increase was blocked when APV was added together with PTX, indicating that the formation of perforated synapses depended on the activation of NMDA receptors. We also showed that inhibition of the tissue type plasminogen activator (tPA-stop/PAI-1) significantly interfered with the activity-induced increase in perforated synapses. This implies that the proteolytic activities of tPA might be involved in steps which are downstream from the NMDA receptor-mediated synaptic plasticity leading to structural changes at synaptic contacts. In contrast, even long-term inhibition of electrical network activity by tetrodotoxin had no effect on the number of perforated synapses, but almost completely abolished the formation of spine synapses. These results indicate that a short-term increase in neural activity via NMDA receptors and a proteolytic cascade involving tPA lead to the formation of perforated synapses.

The weaver mouse gain-of-function phenotype of dopaminergic midbrain neurons is determined by coactivation of wvGirk2 and K-ATP channels.

The phenotype of substantia nigra (SN) neurons in homozygous weaver (wv/wv) mice was studied by combining patch-clamp and single-cell RT-multiplex PCR techniques in midbrain slices of 14-d-old mice. In contrast to GABAergic SN neurons, which were unaffected in homozygous weaver mice (wv/wv), dopaminergic SN neurons possessed a dramatically altered phenotype with a depolarized membrane potential and complete loss of spontaneous pacemaker activity. The gain-of-function phenotype was mediated by a large, nonselective membrane conductance exclusively present in (wv/wv) dopaminergic SN neurons. This constitutively activated conductance displayed a sensitivity to external QX-314 (IC(50) = 10.6 microM) very similar to that of heterologously expressed wvGirk2 channels and was not further activated by G-protein stimulation. Single-cell Girk1-4 expression profiling suggested that homomeric Girk2 channels were present in most dopaminergic SN neurons, whereas Girk2 was always coexpressed with other Girk family members in GABAergic SN neurons. Surprisingly, acute QX-314 inhibition of wvGirk2 channels did not induce wild-type-like pacemaker activity but instead caused membrane hyperpolarization. Additional application of a blocker of ATP-sensitive potassium channels (100 microM tolbutamide) induced wild-type-like pacemaker activity. We conclude that the gain-of-function weaver phenotype of dopaminergic substantia nigra neurons is mediated by coactivation of wvGirk2 and SUR1/Kir6. 2-mediated ATP-sensitive K(+) channels. We also show that in contrast to wild-type neurons, all (wv/wv) dopaminergic SN neurons expressed calbindin, a calcium-binding protein that marks dopaminergic SN neurons resistant to neurodegeneration. The identification of two ion channels that in concert determine the weaver phenotype of surviving calbindin-positive dopaminergic SN neurons will help to understand the molecular mechanisms of selective neurodegeneration of dopaminergic SN neurons in the weaver mouse and might be important in Parkinson's disease.

Functional and molecular aspects of voltage-gated K+ channel beta subunits.

Voltage-gated potassium channels (Kv) of the Shaker-related superfamily are assembled from membrane-integrated alpha subunits and auxiliary beta subunits. The beta subunits may increase Kv channel surface expression and/or confer A-type behavior to noninactivating Kv channels in heterologous expression systems. The interaction of Kv alpha and Kv beta subunits depends on the presence or absence of several domains including the amino-terminal N-type inactivating and NIP domains and the Kv alpha and Kv beta binding domains. Loss of function of Kv beta 1.1 subunits leads to a reduction of A-type Kv channel activity in hippocampal and striatal neurons of knock-out mice. This reduction may be correlated with altered cognition and motor control in the knock-out mice.

Alternative sulfonylurea receptor expression defines metabolic sensitivity of K-ATP channels in dopaminergic midbrain neurons.

ATP-sensitive potassium (K-ATP) channels couple the metabolic state to cellular excitability in various tissues. Several isoforms of the K-ATP channel subunits, the sulfonylurea receptor (SUR) and inwardly rectifying K channel (Kir6.X), have been cloned, but the molecular composition and functional diversity of native neuronal K-ATP channels remain unresolved. We combined functional analysis of K-ATP channels with expression profiling of K-ATP subunits at the level of single substantia nigra (SN) neurons in mouse brain slices using an RT-multiplex PCR protocol. In contrast to GABAergic neurons, single dopaminergic SN neurons displayed alternative co-expression of either SUR1, SUR2B or both SUR isoforms with Kir6.2. Dopaminergic SN neurons expressed alternative K-ATP channel species distinguished by significant differences in sulfonylurea affinity and metabolic sensitivity. In single dopaminergic SN neurons, co-expression of SUR1 + Kir6.2, but not of SUR2B + Kir6.2, correlated with functional K-ATP channels highly sensitive to metabolic inhibition. In contrast to wild-type, surviving dopaminergic SN neurons of homozygous weaver mouse exclusively expressed SUR1 + Kir6.2 during the active period of dopaminergic neurodegeneration. Therefore, alternative expression of K-ATP channel subunits defines the differential response to metabolic stress and constitutes a novel candidate mechanism for the differential vulnerability of dopaminergic neurons in response to respiratory chain dysfunction in Parkinson's disease.

Cloning and functional expression of rat ether-à-go-go-like K+ channel genes.

1. Screening of rat cortex cDNA resulted in cloning of two complete and one partial orthologue of the Drosophila ether-à-go-go-like K+ channel (elk). 2. Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed predominant expression of rat elk mRNAs in brain. Each rat elk mRNA showed a distinct, but overlapping expression pattern in different rat brain areas. 3. Transient transfection of Chinese hamster ovary (CHO) cells with rat elk1 or rat elk2 cDNA gave rise to voltage-activated K+ channels with novel properties. 4. RELK1 channels mediated slowly activating sustained potassium currents. The threshold for activation was at -90 mV. Currents were insensitive to tetraethylammonium (TEA) and 4-aminopyridine (4-AP), but were blocked by micromolar concentrations of Ba2+. RELK1 activation kinetics were not dependent on prepulse potential like REAG-mediated currents. 5. RELK2 channels produced currents with a fast inactivation component and HERG-like tail currents. RELK2 currents were not sensitive to the HERG channel blocker E4031.

NIP domain prevents N-type inactivation in voltage-gated potassium channels.

Shaker-related voltage-gated K+ (Kv) channels are assembled from ion-conducting K(v)alpha subunits, which are integral membrane proteins, and auxiliary K(v)beta subunits. This leads to the formation of highly diverse heteromultimeric Kv channels that mediate outward currents with a wide range of time courses for inactivation. Two principal inactivation mechanisms have been recognized: C-type inactivation correlated with carboxy-terminal K(v)alpha-subunit structures, and N-type inactivation conferred by 'ball' domains in the amino termini of certain K(v)alpha and K(v)beta subunits. Assembly of heteromultimers with one or more K(v)alpha- and/or K(v)beta ball domains appears to be an essential principle of the generation of A-type Kv channel diversity. Here we show that, unexpectedly, the presence of K(v)alpha- or K(v)beta-ball domains does not dominate the gating phenotype in heteromultimers containing Kv1.6alpha subunits. These heteromultimers mediate non-inactivating currents because of the dominant-negative activity of a new type of N-type inactivation-prevention (NIP) domain present in the Kv1.6 amino terminus. Mutations in the NIP domain lead to loss of function, and its transfer to another K(v)alpha subunit leads to gain of function. Our discovery of the NIP domain, which neutralizes the activity of K(v)alpha- and K(v)beta-inactivation gates, establishes a new determinant for the gating behaviour of heteromultimeric Kv channels.

Frequency-dependent inactivation of mammalian A-type K+ channel KV1.4 regulated by Ca2+/calmodulin-dependent protein kinase.

Ca2+/calmodulin dependent protein kinase (CaMKII) and protein phosphatase 2B (calcineurin) are key enzymes in the regulation of synaptic strength, controlling the phosphorylation status of pre- and postsynaptic target proteins. Here, we show that the inactivation gating of the Shaker-related fast-inactivating KV channel, Kv1.4 is controlled by CaMKII and the calcineurin/inhibitor-1 protein phosphatase cascade. CaMKII phosphorylation of an amino-terminal residue of KV1.4 leads to slowing of inactivation gating and accelerated recovery from N-type inactivated states. In contrast, dephosphorylation of this residue induces a fast inactivating mode of KV1.4 with time constants of inactivation 5 to 10 times faster compared with the CaMKII-phosphorylated form. Dephosphorylated KV1.4 channels also display slowed and partial recovery from inactivation with increased trapping of KV1.4 channels in long-absorbing C-type inactivated states. In consequence, dephosphorylated KV1.4 displays a markedly increased tendency to undergo cumulative inactivation during repetitive stimulation. The balance between phosphorylated and dephosphorylated KV1.4 channels is regulated by changes in intracellular Ca2+ concentration rendering KV1.4 inactivation gating Ca2+-sensitive. The reciprocal CaMKII and calcineurin regulation of cumulative inactivation of presynaptic KV1.4 may provide a novel mechanism to regulate the critical frequency for presynaptic spike broadening and induction of synaptic plasticity.

Presynaptic potassium channels.

The past year has witnessed some significant improvements in our understanding of the molecular diversity, subunit composition, and functional properties of K+ channels in heterologous expression systems. Immunocytochemical studies have yielded important information on the localization of K+ channel proteins to synaptic terminals in mammalian brain. Although a coherent picture of native presynaptic K+ channels' function in the mammalian central nervous system is not yet available, it may emerge from improvements in patch-clamp techniques and new applications of targeted knock-out technologies.

Kv beta 1 subunit binding specific for shaker-related potassium channel alpha subunits.

Voltage-activated potassium (Kv) channels from mammalian brain are hetero-oligomers containing alpha and beta subunits. Coexpression of Kv1 alpha and Kv beta 1 subunits confers rapid A-type inactivation on noninactivating potassium channels (delayed rectifiers) in expression systems in vitro. We have delineated a Kv1.5 aminoterminal region of up to 90 amino acids (residues 112-201) that is sufficient for interactions of Kv1.5 alpha and Kv beta 1 subunits. Within this region of the Kv1.5 amino terminus (residues 193-201), a Kv beta 1 interaction site necessary for Kv beta 1-mediated rapid inactivation of Kv1.5 currents was detected. This interaction site motif (FYE/QLGE/DEAM/L) is found exclusively in the Shaker-related subfamily (Kv1). The results show that hetero-oligomerization between alpha and Kv beta 1 subunits is restricted to Shaker-related potassium channel alpha subunits.

Structural and functional characterization of human potassium channel subunit beta 1 (KCNA1B).

Voltage-activated Shaker-related potassium channels (kv1) consist of alpha and beta subunits. We have analysed the structure of the human KCNA1B (hKv beta 1) gene. KCNA1B is > 250 kb in size and encodes at least three Kv beta 1 splice variants. The Kv beta 1 open reading frame is divided into 14 exons. In contrast, genes coding for family members of KCNA (Kv 1 alpha) subunits are markedly smaller and have intronless open reading frames. The expression of Kv 1 alpha and Kv beta mRNA was compared in Northern blots of poly(A+) RNA isolated from various human brain tissues. The results suggest an intricate and cell-specific regulation of Kv 1 alpha and Kv beta mRNA synthesis such that distinct combinations of alpha and beta subunits would occur in different nuclei of the brain. The splice variants hKv beta 1.1 and hKv beta 1.2 were functionally characterized in coexpression studies with hKv 1.5 alpha subunits in 293 cells. It is shown that the confer rapid inactivation on hKv 1.5 channels with different potencies. This may be due to differences in their amino terminal sequences and/or inactivating domains. It is also shown that the amino terminal Kv beta 1.1 and Kv 1.4 alpha inactivating domains compete with each other, probably for the binding to the same receptor site(s) on Kv 1 alpha-subunits.