Bridging vascular physiology to vascular medicine: an integrative laboratory class.

Vascular diseases of the legs are highly prevalent and constitute an important part of medical curricula. The understanding of these diseases relies on strongly interwoven aspects of vascular physiology and vascular medicine. We aimed to connect these within a horizontally integrated laboratory class on vascular physiology of the leg that was designed in cooperation between the departments of physiology and vascular surgery. Conceptually, we applied examination techniques of vascular medicine to visualize physiological parameters that are altered by the most frequent diseases. This facilitates integrative discussions on malfunctions, trains diagnostic skills, and bridges to vascular medicine. In four experiments, we use oscillometry and impedance venous occlusion plethysmography to address key aspects of the arterial and venous system of the legs: 1) arterial pulse wave, 2) arterial systolic blood pressure, 3) venous capacitance and venous outflow, and 4) reactive hyperemia. After the experiments, physiological vascular function, the associated diseases, their impact on the recorded parameters, and diagnostic options are discussed. To allow reproduction, we describe the course structure and the experimental setup in detail. We present the experimental data of a cohort of medical students and document learning success and student satisfaction. All experiments were feasible and provided robust data on physiologically and clinically relevant vascular functions. The activity was perceived positively by the students and led to a substantial improvement of knowledge. With this work, we offer a template for reproduction or variation of a proven concept of horizontally integrated teaching of vascular physiology of the leg.NEW & NOTEWORTHY This article presents an integrative laboratory class on vascular physiology bridging to vascular medicine. The four experiments rely on oscillometry and venous occlusion plethysmography. We describe in detail this new class regarding structure, experimental setup, and experimental procedure, and we give insight into the applied materials. Moreover, we present the experimental data of 74 students and a quantitative evaluation of the students' learning success and acceptance.

KCND2 variants associated with global developmental delay differentially impair Kv4.2 channel gating.

Here, we report on six unrelated individuals, all presenting with early-onset global developmental delay, associated with impaired motor, speech and cognitive development, partly with developmental epileptic encephalopathy and physical dysmorphisms. All individuals carry heterozygous missense variants of KCND2, which encodes the voltage-gated potassium (Kv) channel α-subunit Kv4.2. The amino acid substitutions associated with the variants, p.(Glu323Lys) (E323K), p.(Pro403Ala) (P403A), p.(Val404Leu) (V404L) and p.(Val404Met) (V404M), affect sites known to be critical for channel gating. To unravel their likely pathogenicity, recombinant mutant channels were studied in the absence and presence of auxiliary ß-subunits under two-electrode voltage clamp in Xenopus oocytes. All channel mutants exhibited slowed and incomplete macroscopic inactivation, and the P403A variant in addition slowed activation. Co-expression of KChIP2 or DPP6 augmented the functional expression of both wild-type and mutant channels; however, the auxiliary ß-subunit-mediated gating modifications differed from wild type and among mutants. To simulate the putative setting in the affected individuals, heteromeric Kv4.2 channels (wild type + mutant) were studied as ternary complexes (containing both KChIP2 and DPP6). In the heteromeric ternary configuration, the E323K variant exhibited only marginal functional alterations compared to homomeric wild-type ternary, compatible with mild loss-of-function. By contrast, the P403A, V404L and V404M variants displayed strong gating impairment in the heteromeric ternary configuration, compatible with loss-of-function or gain-of-function. Our results support the etiological involvement of Kv4.2 channel gating impairment in early-onset monogenic global developmental delay. In addition, they suggest that gain-of-function mechanisms associated with a substitution of V404 increase epileptic seizure susceptibility.

Modulation of Kv4.2/KChIP3 interaction by the ceroid lipofuscinosis neuronal 3 protein CLN3.

Voltage-gated potassium (Kv) channels of the Kv4 subfamily associate with Kv channel-interacting proteins (KChIPs), which leads to enhanced surface expression and shapes the inactivation gating of these channels. KChIP3 has been reported to also interact with the late endosomal/lysosomal membrane glycoprotein CLN3 (ceroid lipofuscinosis neuronal 3), which is modified because of gene mutation in juvenile neuronal ceroid lipofuscinosis (JNCL). The present study was undertaken to find out whether and how CLN3, by its interaction with KChIP3, may indirectly modulate Kv4.2 channel expression and function. To this end, we expressed KChIP3 and CLN3, either individually or simultaneously, together with Kv4.2 in HEK 293 cells. We performed co-immunoprecipitation experiments and found a lower amount of KChIP3 bound to Kv4.2 in the presence of CLN3. In whole-cell patch-clamp experiments, we examined the effects of CLN3 co-expression on the KChIP3-mediated modulation of Kv4.2 channels. Simultaneous co-expression of CLN3 and KChIP3 with Kv4.2 resulted in a suppression of the typical KChIP3-mediated modulation; i.e. we observed less increase in current density, less slowing of macroscopic current decay, less acceleration of recovery from inactivation, and a less positively shifted voltage dependence of steady-state inactivation. The suppression of the KChIP3-mediated modulation of Kv4.2 channels was weaker for the JNCL-related missense mutant CLN3R334C and for a JNCL-related C-terminal deletion mutant (CLN3ΔC). Our data support the notion that CLN3 is involved in Kv4.2/KChIP3 somatodendritic A-type channel formation, trafficking, and function, a feature that may be lost in JNCL.

Kv channel-interacting proteins as neuronal and non-neuronal calcium sensors.

Kv channel-interacting proteins (KChIPs) belong to the neuronal calcium sensor (NCS) family of Ca2+-binding EF-hand proteins. KChIPs constitute a group of specific auxiliary ß-subunits for Kv4 channels, the molecular substrate of transient potassium currents in both neuronal and non-neuronal tissues. Moreover, KChIPs can interact with presenilins to control ER calcium signaling and apoptosis, and with DNA to control gene transcription. Ca2+ binding via their EF-hands, with the consequence of conformationl changes, is well documented for KChIPs. Moreover, the Ca2+ dependence of the presenilin/KChIP complex may be related to Alzheimer's disease and the Ca2+ dependence of the DNA/KChIP complex to pain sensing. However, only in few cases could the Ca2+ binding to KChIPs be directly linked to the control of excitability in nerve and muscle cells known to express Kv4/KChIP channel complexes. This review summarizes current knowledge about the Ca2+ binding properties of KChIPs and the Ca2+ dependencies of macromolecular complexes containing KChIPs, including those with presenilins, DNA and especially Kv4 channels. The respective physiological or pathophysiolgical roles of Ca2+ binding to KChIPs are discussed.

Somatodendritic surface expression of epitope-tagged and KChIP binding-deficient Kv4.2 channels in hippocampal neurons.

Kv4.2 channels mediate a subthreshold-activating somatodendritic A-type current (ISA) in hippocampal neurons. We examined the role of accessory Kv channel interacting protein (KChIP) binding in somatodendritic surface expression and activity-dependent decrease in the availability of Kv4.2 channels. For this purpose we transfected cultured hippocampal neurons with cDNA coding for Kv4.2 wild-type (wt) or KChIP binding-deficient Kv4.2 mutants. All channels were equipped with an externally accessible hemagglutinin (HA)-tag and an EGFP-tag, which was attached to the C-terminal end. Combined analyses of EGFP self-fluorescence, surface HA immunostaining and patch-clamp recordings demonstrated similar dendritic trafficking and functional surface expression for Kv4.2[wt]HA,EGFP and the KChIP binding-deficient Kv4.2[A14K]HA,EGFP. Coexpression of exogenous KChIP2 augmented the surface expression of Kv4.2[wt]HA,EGFP but not Kv4.2[A14K]HA,EGFP. Notably, activity-dependent decrease in availability was more pronounced in Kv4.2[wt]HA,EGFP + KChIP2 coexpressing than in Kv4.2[A14K]HA,EGFP + KChIP2 coexpressing neurons. Our results do not support the notion that accessory KChIP binding is a prerequisite for dendritic trafficking and functional surface expression of Kv4.2 channels, however, accessory KChIP binding may play a potential role in Kv4.2 modulation during intrinsic plasticity processes.

Modulation of human Kv4.3/KChIP2 channel inactivation kinetics by cytoplasmic Ca2.

The transient outward current (I to) in the human heart is mediated by Kv4.3 channels complexed with Kv channel interacting protein (KChIP) 2, a cytoplasmic Ca2+-binding EF-hand protein known to modulate Kv4.3 inactivation gating upon heterologous co-expression. We studied Kv4.3 channels co-expressed with wild-type (wt) or EF-hand-mutated (ΔEF) KChIP2 in human embryonic kidney (HEK) 293 cells. Co-expression took place in the absence or presence of BAPTA-AM, and macroscopic currents were recorded in the whole-cell patch-clamp configuration with different free Ca2+ concentrations in the patch-pipette. Our data indicate that Ca2+ is not necessary for Kv4.3/KChIP2 complex formation. The Kv4.3/KChIP2-mediated current decay was faster and the recovery of Kv4.3/KChIP2 channels from inactivation slower with 50 µM Ca2+ than with BAPTA (nominal Ca2+-free) in the patch-pipette. The apparent Ca2+-mediated slowing of recovery kinetics was still observed when EF-hand 4 of KChIP2 was mutated (ΔEF4) but not when EF-hand 2 (ΔEF2) was mutated, and turned into a Ca2+-mediated acceleration of recovery kinetics when EF-hand 3 (ΔEF3) was mutated. In the presence of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 cytoplasmic Ca2+ (50 µM) induced an acceleration of Kv4.3/KChIP2 recovery kinetics, which was still observed when EF-hand 2 was mutated (ΔEF2) but not when EF-hand 3 (ΔEF3) or EF-hand 4 (ΔEF4) was mutated. Our results support the notion that binding of Ca2+ to KChIP2 EF-hands can acutely modulate Kv4.3/KChIP2 channel inactivation gating, but the Ca2+-dependent gating modulation depends on CaMKII action. Our findings speak for an acute modulation of I to kinetics and frequency-dependent I to availability in cardiomyocytes under conditions with elevated Ca2+ levels and CaMKII activity.

KChIP2 genotype dependence of transient outward current (Ito) properties in cardiomyocytes isolated from male and female mice.

The transient outward current (Ito) in cardiomyocytes is largely mediated by Kv4 channels associated with Kv Channel Interacting Protein 2 (KChIP2). A knockout model has documented the critical role of KChIP2 in Ito expression. The present study was conducted to characterize in both sexes the dependence of Ito properties, including current magnitude, inactivation kinetics, recovery from inactivation and voltage dependence of inactivation, on the number of functional KChIP2 alleles. For this purpose we performed whole-cell patch-clamp experiments on isolated left ventricular cardiomyocytes from male and female mice which had different KChIP2 genotypes; i.e., wild-type (KChIP2+/+), heterozygous knockout (KChIP2+/-) or complete knockout of KChIP2 (KChIP2-/-). We found in both sexes a KChIP2 gene dosage effect (i.e., a proportionality between number of alleles and phenotype) on Ito magnitude, however, concerning other Ito properties, KChIP2+/- resembled KChIP2+/+. Only in the total absence of KChIP2 (KChIP2-/-) we observed a slowing of Ito kinetics, a slowing of recovery from inactivation and a negative shift of a portion of the voltage dependence of inactivation. In a minor fraction of KChIP2-/- myocytes Ito was completely lost. The distinct KChIP2 genotype dependences of Ito magnitude and inactivation kinetics, respectively, seen in cardiomyocytes were reproduced with two-electrode voltage-clamp experiments on Xenopus oocytes expressing Kv4.2 and different amounts of KChIP2. Our results corroborate the critical role of KChIP2 in controlling Ito properties. They demonstrate that the Kv4.2/KChIP2 interaction in cardiomyocytes is highly dynamic, with a clear KChIP2 gene dosage effect on Kv4 channel surface expression but not on inactivation gating.

Marine compound rhizochalinin shows high in vitro and in vivo efficacy in castration resistant prostate cancer.

Development of drug resistance is an inevitable phenomenon in castration-resistant prostate cancer (CRPC) cells requiring novel therapeutic approaches. In this study, efficacy and toxicity of Rhizochalinin (Rhiz) - a novel sphingolipid-like marine compound - was evaluated in prostate cancer models, resistant to currently approved standard therapies. In vitro activity and mechanism of action of Rhiz were examined in the human prostate cancer cell lines PC-3, DU145, LNCaP, 22Rv1, and VCaP. Rhiz significantly reduced cell viability at low micromolar concentrations showing most pronounced effects in enzalutamide and abiraterone resistant AR-V7 positive cells. Caspase-dependent apoptosis, inhibition of pro-survival autophagy, downregulation of AR-V7, PSA and IGF-1 expression as well as inhibition of voltage-gated potassium channels were identified as mechanisms of action. Remarkably, Rhiz re-sensitized AR-V7 positive cells to enzalutamide and increased efficacy of taxanes.In vivo activity and toxicity were evaluated in PC-3 and 22Rv1 NOD SCID mouse xenograft models using i.p. administration. Rhiz significantly reduced growth of PC-3 and 22Rv1 tumor xenografts by 27.0% (p = 0.0156) and 46.8% (p = 0.047) compared with controls with an increased fraction of tumor cells showing apoptosis secondary to Rhiz exposure. In line with the in vitro data, Rhiz was most active in AR-V7 positive xenografts in vivo. In animals, no severe side effects were observed.In conclusion, Rhiz is a promising novel marine-derived compound characterized by a unique combination of anticancer properties. Its further clinical development is of high impact for patients suffering from drug resistant prostate cancer especially those harboring AR-V7 mediated resistance to enzalutamide and abiraterone.

Intra- and Intersubunit Dynamic Binding in Kv4.2 Channel Closed-State Inactivation.

We studied the kinetics and structural determinants of closed-state inactivation (CSI) in Kv4.2 channels, considering a multistep process and the possibility that both intra- and intersubunit dynamic binding (i.e., loss and restoration of physical contact) may occur between the S4-S5 linker, including the initial S5 segment (S4S5), and the S6 gate. We expressed Kv4.2 channels in Xenopus oocytes and measured the onset of low-voltage inactivation under two-electrode voltage clamp. Indicative of a transitory state, the onset kinetics were best described by a double-exponential function. To examine the involvement of individual S4S5 and S6 amino acid residues in dynamic binding, we studied S4S5 and S6 single alanine mutants and corresponding double mutants. Both transitory and steady-state inactivation were modified by these mutations, and we quantified the mutational effects based on apparent affinities for the respective inactivated states. Double-mutant cycle analyses revealed strong functional coupling of the S6 residues V404 and I412 to all tested S4S5 residues. To examine whether dynamic S4S5/S6 binding occurs within individual α-subunits or between neighboring α-subunits, we performed a double-mutant cycle analysis with Kv4.2 tandem-dimer constructs. The constructs carried either an S4S5/S6 double mutation in the first α-subunit and no mutation in the second (concatenated) α-subunit or an S4S5 point mutation in the first α-subunit and an S6 point mutation in the second α-subunit. Our results support the notion that CSI in Kv4.2 channels is a multistep process that involves dynamic binding both within individual α-subunits and between neighboring α-subunits.

Easy method to examine single nerve fiber excitability and conduction parameters using intact nonanesthetized earthworms.

The generation and conduction of neuronal action potentials (APs) were the subjects of a cell physiology exercise for first-year medical students. In this activity, students demonstrated the all-or-none nature of AP generation, measured conduction velocity, and examined the dependence of the threshold stimulus amplitude on stimulus duration. For this purpose, they used the median giant nerve fiber (MGF) in the ventral nerve cord of the common earthworm (Lumbricus terrestris). Here, we introduce a specialized stimulation and recording chamber that the nonanesthetized earthworm enters completely unforced. The worm resides in a narrow round duct with silver electrodes on the bottom such that individual APs of the MGF can be elicited and recorded superficially. Our experimental setup combines several advantages: it allows noninvasive single fiber AP measurements taken from a nonanesthetized animal that is yet restrained. Students performed the experiments with a high success rate. According to the data acquired by the students, the mean conduction velocity of the MGF was 30.2 m/s. From the amplitude-duration relationship for threshold stimulation, rheobase and chronaxie were graphically determined by the students according to Lapicque's method. The mean rheobase was 1.01 V, and the mean chronaxie was 0.06 ms. The acquired data and analysis results are of high quality, as deduced from critical examination based on the law of Weiss. In addition, we provide video material, which was also used in the practical course.

Nedd4-2 regulates surface expression and may affect N-glycosylation of hyperpolarization-activated cyclic nucleotide-gated (HCN)-1 channels.

HCN channels are important regulators of neuronal excitability. The proper function of these channels is governed by various mechanisms, including post-translational modifications of channel subunits. Here, we provide evidence that ubiquitination via a ubiquitin ligase, neuronal precursor cell expressed developmentally downregulated (Nedd)-4-2, is involved in the regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We identified a PY motif (L/PPxY), the characteristic binding motif for Nedd4-2 in the C terminus of the HCN1 subunit, and showed that HCN1 and Nedd4-2 interacted both in vivo (rat hippocampus, neocortex, and cerebellum) and in vitro [human embryonic kidney 293 (HEK293) cells], resulting in increased HCN1 ubiquitination. Elimination of the PY motif reduced, but did not abolish, Nedd4-2 binding, which further involved a stretch of ∼100 aa downstream in the HCN1 C terminus. Coexpression of Nedd4-2 and HCN1 drastically reduced the HCN1-mediated h-current amplitude (85-92%) in Xenopus laevis oocytes and reduced surface expression (34%) of HCN1 channels in HEK293 cells, thereby opposing effects of tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b)-(1a-4), an auxiliary subunit that promotes HCN1 surface expression. Regulation may further include N-glycosylation of HCN1 channels, which is significantly enhanced by TRIP8b(1a-4), but may be reduced by Nedd4-2. Taken together, our data indicate that Nedd4-2 plays an important role in the regulation of HCN1 trafficking and may compete with TRIP8b(1a-4) in this process.

Hippocampal A-type current and Kv4.2 channel modulation by the sulfonylurea compound NS5806.

We examined the effects of the sulfonylurea compound NS5806 on neuronal A-type channel function. Using whole-cell patch-clamp we studied the effects of NS5806 on the somatodendritic A-type current (I(SA)) in cultured hippocampal neurons and the currents mediated by Kv4.2 channels coexpressed with different auxiliary ß-subunits, including both Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-related proteins (DPPs), in HEK 293 cells. The amplitude of the I(SA) component in hippocampal neurons was reduced in the presence of 20 µM NS5806. I(SA) decay kinetics were slowed and the recovery kinetics accelerated, but the voltage dependence of steady-state inactivation was shifted to more negative potentials by NS5806. The peak amplitudes of currents mediated by ternary Kv4.2 channel complexes, associated with DPP6-S (short splice-variant) and either KChIP2, KChIP3 or KChIP4, were potentiated and their macroscopic inactivation slowed by NS5806, whereas the currents mediated by binary Kv4.2 channels, associated only with DPP6-S, were suppressed, and the NS5806-mediated slowing of macroscopic inactivation was less pronounced. Neither potentiation nor suppression and no effect on current decay kinetics in the presence of NS5806 were observed for Kv4.2 channels associated with KChIP3 and the N-type inactivation-conferring DPP6a splice-variant. For all recombinant channel complexes, NS5806 slowed the recovery from inactivation and shifted the voltage dependence of steady-state inactivation to more negative potentials. Our results demonstrate the activity of NS5806 on native I(SA) and possible molecular correlates in the form of recombinant Kv4.2 channels complexed with different KChIPs and DPPs, and they shed some light on the mechanism of NS5806 action.

Voltage sensor inactivation in potassium channels.

In voltage-gated potassium (Kv) channels membrane depolarization causes movement of a voltage sensor domain. This conformational change of the protein is transmitted to the pore domain and eventually leads to pore opening. However, the voltage sensor domain may interact with two distinct gates in the pore domain: the activation gate (A-gate), involving the cytoplasmic S6 bundle crossing, and the pore gate (P-gate), located externally in the selectivity filter. How the voltage sensor moves and how tightly it interacts with these two gates on its way to adopt a relaxed conformation when the membrane is depolarized may critically determine the mode of Kv channel inactivation. In certain Kv channels, voltage sensor movement leads to a tight interaction with the P-gate, which may cause conformational changes that render the selectivity filter non-conductive ("P/C-type inactivation"). Other Kv channels may preferably undergo inactivation from pre-open closed-states during voltage sensor movement, because the voltage sensor temporarily uncouples from the A-gate. For this behavior, known as "preferential" closed-state inactivation, we introduce the term "A/C-type inactivation". Mechanistically, P/C- and A/C-type inactivation represent two forms of "voltage sensor inactivation."

Inositol-1,4,5-trisphosphate 3-kinase A regulates dendritic morphology and shapes synaptic Ca2+ transients.

Inositol-1,4,5-trisphosphate 3-kinase-A (itpka) accumulates in dendritic spines and seems to be critically involved in synaptic plasticity. The protein possesses two functional activities: it phosphorylates inositol-1,4,5-trisphosphate (Ins(1,4,5)P(3)) and regulates actin dynamics by its F-actin bundling activity. To assess the relevance of these activities for neuronal physiology, we examined the effects of altered itpka levels on cell morphology, Ins(1,4,5)P(3) metabolism and dendritic Ca(2+) signaling in hippocampal neurons. Overexpression of itpka increased the number of dendritic protrusions by 71% in immature primary neurons. In mature neurons, however, the effect of itpka overexpression on formation of dendritic spines was weaker and depletion of itpka did not alter spine density and synaptic contacts. In synaptosomes of mature neurons itpka loss resulted in decreased duration of Ins(1,4,5)P(3) signals and shorter Ins(1,4,5)P(3)-dependent Ca(2+) transients. At synapses of itpka deficient neurons the levels of Ins(1,4,5)P(3)-5-phosphatase (inpp5a) and sarcoplasmic/endoplasmic reticulum calcium ATPase pump-2b (serca2b) were increased, indicating that decreased duration of Ins(1,4,5)P(3) and Ca(2+) signals results from compensatory up-regulation of these proteins. Taken together, our data suggest a dual role for itpka. In developing neurons itpka has a morphogenic effect on dendrites, while the kinase appears to play a key role in shaping Ca(2+) transients at mature synapses.

Acute alterations of somatodendritic action potential dynamics in hippocampal CA1 pyramidal cells after kainate-induced status epilepticus in mice.

Pathophysiological remodeling processes at an early stage of an acquired epilepsy are critical but not well understood. Therefore, we examined acute changes in action potential (AP) dynamics immediately following status epilepticus (SE) in mice. SE was induced by intraperitoneal (i.p.) injection of kainate, and behavioral manifestation of SE was monitored for 3-4 h. After this time interval CA1 pyramidal cells were studied ex vivo with whole-cell current-clamp and Ca(2+) imaging techniques in a hippocampal slice preparation. Following acute SE both resting potential and firing threshold were modestly depolarized (2-5 mV). No changes were seen in input resistance or membrane time constant, but AP latency was prolonged and AP upstroke velocity reduced following acute SE. All cells showed an increase in AP halfwidth and regular (rather than burst) firing, and in a fraction of cells the notch, typically preceding spike afterdepolarization (ADP), was absent following acute SE. Notably, the typical attenuation of backpropagating action potential (b-AP)-induced Ca(2+) signals along the apical dendrite was strengthened following acute SE. The effects of acute SE on the retrograde spread of excitation were mimicked by applying the Kv4 current potentiating drug NS5806. Our data unveil a reduced somatodendritic excitability in hippocampal CA1 pyramidal cells immediately after acute SE with a possible involvement of both Na(+) and K(+) current components.

Muskelin regulates actin filament- and microtubule-based GABA(A) receptor transport in neurons.

Intracellular transport regulates protein turnover including endocytosis. Because of the spatial segregation of F-actin and microtubules, internalized cargo vesicles need to employ myosin and dynein motors to traverse both cytoskeletal compartments. Factors specifying cargo delivery across both tracks remain unknown. We identified muskelin to interconnect retrograde F-actin- and microtubule-dependent GABA(A) receptor (GABA(A)R) trafficking. GABA(A)Rs regulate synaptic transmission, plasticity, and network oscillations. GABA(A)R α1 and muskelin interact directly, undergo neuronal cotransport, and associate with myosin VI or dynein motor complexes in subsequent steps of GABA(A)R endocytosis. Inhibition of either transport route selectively interferes with receptor internalization or degradation. Newly generated muskelin KO mice display depletion of both transport steps and a high-frequency ripple oscillation phenotype. A diluted coat color of muskelin KOs further suggests muskelin transport functions beyond neurons. Our data suggest the concept that specific trafficking factors help cargoes to traverse both F-actin and microtubule compartments, thereby regulating their fate.

Mechanisms of closed-state inactivation in voltage-gated ion channels.

Inactivation of voltage-gated ion channels is an intrinsic auto-regulatory process necessary to govern the occurrence and shape of action potentials and establish firing patterns in excitable tissues. Inactivation may occur from the open state (open-state inactivation, OSI) at strongly depolarized membrane potentials, or from pre-open closed states (closed-state inactivation, CSI) at hyperpolarized and modestly depolarized membrane potentials. Voltage-gated Na(+), K(+), Ca(2+) and non-selective cationic channels utilize both OSI and CSI. Whereas there are detailed mechanistic descriptions of OSI, much less is known about the molecular basis of CSI. Here, we review evidence for CSI in voltage-gated cationic channels (VGCCs) and recent findings that shed light on the molecular mechanisms of CSI in voltage-gated K(+) (Kv) channels. Particularly, complementary observations suggest that the S4 voltage sensor, the S4S5 linker and the main S6 activation gate are instrumental in the installment of CSI in Kv4 channels. According to this hypothesis, the voltage sensor may adopt a distinct conformation to drive CSI and, depending on the stability of the interactions between the voltage sensor and the pore domain, a closed-inactivated state results from rearrangements in the selectivity filter or failure of the activation gate to open. Kv4 channel CSI may efficiently exploit the dynamics of the subthreshold membrane potential to regulate spiking properties in excitable tissues.

Functional consequences of the interactions among the neural cell adhesion molecule NCAM, the receptor tyrosine kinase TrkB, and the inwardly rectifying K+ channel KIR3.3.

Cell adhesion molecules and neurotrophin receptors are crucial for the development and the function of the nervous system. Among downstream effectors of neurotrophin receptors and recognition molecules are ion channels. Here, we provide evidence that G protein-coupled inwardly rectifying K(+) channel Kir3.3 directly binds to the neural cell adhesion molecule (NCAM) and neurotrophin receptor TrkB. We identified the binding sites for NCAM and TrkB at the C-terminal intracellular domain of Kir3.3. The interaction between NCAM, TrkB, and Kir3.3 was supported by immunocytochemical co-localization of Kir3.3, NCAM, and/or TrkB at the surface of hippocampal neurons. Co-expression of TrkB and Kir3.1/3.3 in Xenopus oocytes increased the K(+) currents evoked by Kir3.1/3.3 channels. This current enhancement was reduced by the concomitant co-expression with NCAM. Both surface fluorescence measurements of microinjected oocytes and cell surface biotinylation of transfected CHO cells indicated that the cell membrane localization of Kir3.3 is regulated by TrkB and NCAM. Furthermore, the level of Kir3.3, but not of Kir3.2, at the plasma membranes was reduced in TrkB-deficient mice, supporting the notion that TrkB regulates the cell surface expression of Kir3.3. The premature expression of developmentally late appearing Kir3.1/3.3 in hippocampal neurons led to a reduction of NCAM-induced neurite outgrowth. Our observations indicate a decisive role for the neuronal K(+) channel in regulating NCAM-dependent neurite outgrowth and attribute a physiologically meaningful role to the functional interplay of Kir3.3, NCAM, and TrkB in ontogeny.

Molecular and functional remodeling of I(to) by angiotensin II in the mouse left ventricle.

The transient outward potassium current (I(to)) in cardiac myocytes is mainly mediated by members of the Kv4 subfamily of voltage-gated potassium channels. Several in vitro studies have shown that angiotensin II (Ang II), which plays an important role in the development of cardiac hypertrophy, rapidly downregulates Kv4.3 mRNA expression. However, it is not clear whether Ang II regulates I(to)in vivo and whether this regulation may depend on alterations in Kv4.3 gene expression. To address this question, we determined the effects of acute (24 h) and chronic (14 days) exogenous infusions of Ang II on I(to) and the expression of its channel subunits in the mouse left ventricle. Ang II rapidly increased blood pressure and reduced Kv4.2 but not Kv4.3 mRNA levels in the absence of cardiac hypertrophy. In response to chronically elevated Ang II levels cardiac hypertrophy developed, which was associated with a downregulation of Kv4.2 and Kv4.3 mRNA levels, and an upregulation of Kv1.4 mRNA levels. In contrast, neither KChIP2 mRNA levels nor amplitude or macroscopic inactivation kinetics of I(to) were affected by the acute or chronic Ang II treatments. Consistent with the unchanged I(to) amplitude, Kv4.2, Kv4.3, and KChIP protein expression levels were similar after chronic Ang II and sham treatment. Our findings demonstrate that elevations of Ang II concentrations that induce hypertension and cardiac hypertrophy do not alter the amplitude of I(to) in the mouse left ventricle. Furthermore, they suggest that functional expression of cardiac I(to) in mice is stabilized by KChIP2.

Characterisation of the R276A gain-of-function mutation in the ectodomain of murine P2X7.

The cytolytic P2X7 purinoceptor is widely expressed on leukocytes and has sparked interest because of its key role in the activation of the inflammasome, the release of the pro-inflammatory cytokine IL-1beta and cell death. We report here the functional characterisation of a R276A gain-of-function mutant analysed for its capacities to induce membrane depolarisation, calcium influx and opening of a large membrane pore permeable to YO-PRO-1. Our results highlight the particular sensitivity of R276A mutant to low micromolar adenosine triphosphate (ATP) concentrations, which possibly reflect an increased affinity for its ligands, and a slower closing kinetics of the receptor channel. Our findings support the notion that evolutionary pressures maintain the low sensitivity of P2X7 to ATP. We also believe that the R276A mutant described here may be useful for the generation of new animal models with exacerbated P2X7 functions that will serve to better characterise its role in inflammation and in immune responses.