Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1_S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1_S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1_L, Thr²⁹. Furthermore, the association of HVCN1_S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1_S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1_S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.The voltage-gated proton channel HVCN1 (or H_V1 or VSOP) is a small protein that conducts protons across membranes selectively (1, 2) and in a regulated manner. Previously, we described its function in B lymphocytes, where proton channels sustain B-cell receptor (BCR) signaling via regulation of reactive oxygen species production by the NADPH oxidase enzyme complex (3). In addition, we found HVCN1 to be directly associated with the BCR. Upon receptor stimulation, the BCR and HVCN1 were cointernalized to late endosomal/lysosomal organelles called “MIICs,” or MHC class II-containing compartments, where antigens bound to the BCR are digested into small peptides and loaded onto MHC class II molecules for presentation to T cells (3).HVCN1 is expressed not only by normal but also by malignant B cells, such as those in chronic lymphocytic leukemia (CLL) (3). CLL cells are characterized by their reliance on BCR signaling for survival and growth (4), so it is possible that they maintain or upregulate HVCN1 expression to sustain their growth. Other tumor cells, such as those in breast (5) and colorectal cancer (6), have been found to rely on HVCN1 for survival. In these tumor cells, proton channels prevent excessive acidification of the cytoplasm and allow increased cell migration. In malignant B cells, HVCN1 may regulate intracellular pH and at the same time sustain BCR signaling. However, its precise roles remain to be elucidated.We show here that CLL cells and other B-cell lines specifically express higher levels of a shorter isoform of HVCN1, HVCN1_S. We identified the existence of two distinct isoforms of relatively similar size when immunoblotting B-cell lysates with an HVCN1-specific antibody (3). HVCN1_S is only weakly expressed in normal B cells, and in light of its apparent upregulation in tumor cells, we set out to characterize its function. We show that HVCN1_S responds more strongly to phosphorylation by protein kinase C (PKC) and identify the phosphorylation site. We provide evidence that HVCN1_S in B cells is preferentially expressed at the plasma membrane, even upon BCR stimulation and subsequent internalization, due to a weaker association with the BCR. Finally, we show that HVCN1_S expression results in stronger BCR signaling, increased proliferation, and augmented chemokine-dependent migration. Overall, our data indicate that HVCN1_S is an alternative protein isoform that mediates stronger currents upon PKC phosphorylation, is more highly expressed at the plasma membrane, and can confer a growth advantage to malignant B cells.     

UDP‐glucose enhances outward K+ currents necessary for cell differentiation and stimulates cell migration by activating the GPR17 receptor in oligodendrocyte precursors

In the developing and mature central nervous system, NG2 expressing cells comprise a population of cycling oligodendrocyte progenitor cells (OPCs) that differentiate into mature, myelinating oligodendrocytes (OLGs). OPCs are also characterized by high motility and respond to injury by migrating into the lesioned area to support remyelination. K⁺ currents in OPCs are developmentally regulated during differentiation. However, the mechanisms regulating these currents at different stages of oligodendrocyte lineage are poorly understood. Here we show that, in cultured primary OPCs, the purinergic G‐protein coupled receptor GPR17, that has recently emerged as a key player in oligodendrogliogenesis, crucially regulates K⁺ currents. Specifically, receptor stimulation by its agonist UDP‐glucose enhances delayed rectifier K⁺ currents without affecting transient K⁺ conductances. This effect was observed in a subpopulation of OPCs and immature pre‐OLGs whereas it was absent in mature OLGs, in line with GPR17 expression, that peaks at intermediate phases of oligodendrocyte differentiation and is thereafter downregulated to allow terminal maturation. The effect of UDP‐glucose on K⁺ currents is concentration‐dependent, blocked by the GPR17 antagonists MRS2179 and cangrelor, and sensitive to the K⁺ channel blocker tetraethyl‐ammonium, which also inhibits oligodendrocyte maturation. We propose that stimulation of K⁺ currents is responsible for GPR17‐induced oligodendrocyte differentiation. Moreover, we demonstrate, for the first time, that GPR17 activation stimulates OPC migration, suggesting an important role for this receptor after brain injury. Our data indicate that modulation of GPR17 may represent a strategy to potentiate the post‐traumatic response of OPCs under demyelinating conditions, such as multiple sclerosis, stroke, and brain trauma.     

和厚朴酚对小鼠背根神经节河豚毒素敏感型钠电流的影响

采用全细胞膜片钳技术,观察和厚朴酚(honokiol,Hnk)对急性分离的小鼠背根神经节上河豚毒素敏感型(TTX-S)钠电流及其通道动力学的影响,探讨Hnk可能的镇痛机制及作用靶点。结果显示:和厚朴酚浓度依赖性地抑制TTX-S钠电流,30 μmol/L和厚朴酚可以使稳态激活曲线向较正电压方向偏移达10.2 mV,通道失活后恢复时间明显延长,但对稳态失活动力学特征无明显的影响。     

Differentiating embryonic stem-derived neural stem cells show a maturation-dependent pattern of voltage-gated sodium current expression and graded action potentials

A population of mouse embryonic stem (ES)-derived neural stem cells (named NS cells) that exhibits traits reminiscent of radial glia-like cell population and that can be homogeneously expanded in monolayer while remaining stable and highly neurogenic over multiple passages has been recently discovered. This novel population has provided a unique in vitro system in which to investigate physiological events occurring as stem cells lose multipotency and terminally differentiate. Here we analysed the timing, quality and quantity of the appearance of the excitability properties of differentiating NS cells which have been long-term expanded in vitro. To this end, we studied the biophysical properties of voltage-dependent Na(+) currents as an electrophysiological readout for neuronal maturation stages of differentiating NS cells toward the generation of fully functional neurons, since the expression of neuronal voltage-gated Na(+) channels is an essential hallmark of neuronal differentiation and crucial for signal transmission in the nervous system. Using the whole cell and single-channel cell-attached variations of the patch-clamp technique we found that the Na(+) currents in NS cells showed substantial electrophysiological changes during in vitro neuronal differentiation, consisting mainly in an increase of Na(+) current density and in a shift of the steady-state activation and inactivation curves toward more negative and more positive potentials respectively. The changes in the Na(+) channel system were closely related with the ability of differentiating NS cells to generate action potentials, and could therefore be exploited as an appropriate electrophysiological marker of ES-derived NS cells undergoing functional neuronal maturation.     

Adenosine A2A receptors and basal ganglia physiology

Adenosine A2A receptors are highly enriched in the basal ganglia system. They are predominantly expressed in enkephalin-expressing GABAergic striatopallidal neurons and therefore are highly relevant to the function of the indirect efferent pathway of the basal ganglia system. In these GABAergic enkephalinergic neurons, the A2A receptor tightly interacts structurally and functionally with the dopamine D2 receptor. Both by forming receptor heteromers and by targeting common intracellular signaling cascades, A2A and D2 receptors exhibit reciprocal antagonistic interactions that are central to the function of the indirect pathway and hence to basal ganglia control of movement, motor learning, motivation and reward. Consequently, this A2A/D2 receptors antagonistic interaction is also central to basal ganglia dysfunction in Parkinson's disease. However, recent evidence demonstrates that, in addition to this post-synaptic site of action, striatal A2A receptors are also expressed and have physiological relevance on pre-synaptic glutamatergic terminals of the cortico-limbic-striatal and thalamo-striatal pathways, where they form heteromeric receptor complexes with adenosine A1 receptors. Therefore, A2A receptors play an important fine-tuning role, boosting the efficiency of glutamatergic information flow in the indirect pathway by exerting control, either pre- and/or post-synaptically, over other key modulators of glutamatergic synapses, including D2 receptors, group I metabotropic mGlu5 glutamate receptors and cannabinoid CB1 receptors, and by triggering the cAMP-protein kinase A signaling cascade.     

Vascular effects of class-III antiarrhythmic drugs: chromanol 293B, but not dofetilide blocks the smooth muscle delayed rectifier K+ channel

Chromanol 293B and dofetilide are inhibitors of IK_s and IK_r, i.e., of the slow and the rapid component of the delayed rectifier potassium current. The specificity of these drugs was tested by investigating their effects on the delayed rectifier potassium current in vascular smooth muscle, regulating the tone of blood vessels. Using depolarizing step protocols with asymmetrical potassium concentrations (135/4.5 mM K⁺ in pipette/bath), voltage-dependent K⁺ currents (IK_v) of enzymatically dispersed guinea pig portal vein cells were studied in the whole-cell patch-clamp technique. Peak currents were obtained within 20 ms (at +50 mV) after activation. During a 10 s test pulse to +60 mV, these currents exhibited a relatively fast inactivation with time constants of 384 ms (τ_fast) and 4505 ms (τ_slow). Dofetilide was totally ineffective in modulating currents; in contrast, after application of chromanol 293B, a steady-state block of IK_v developed within 135 s. The block was concentration-dependent with an IC₅₀ of 7.4 μM. Chromanol did not produce any shift in the normalized steady-state activation and inactivation curves and the recovery from inactivation was not significantly changed. Chromanol 293B similarly inhibited delayed rectifier K⁺ channels whether in their closed or open state, and produced an “apparent” acceleration of inactivation, i.e., the drug accelerated the faster time constant of inactivation during a 10 s test pulse from 384 ms (control) to 149 ms (100 μM chromanol). In recent studies, chromanol was described as a specific blocker of slowly activating delayed rectifier potassium channels (IK_s) in cardiomyocytes. The results of this study, however, extend the inhibitory spectrum of the drug and demonstrate block of closed and open state delayed rectifier K⁺ currents in portal vein vascular smooth muscle. Such a block could possibly contribute to the generation of portal hypertension. Received: 2 March 2001, Returned for 1. revision: 22 March 2001, 1. Revision received: 9 May 2001, Returned for 2. revision: 16 May 2001, 2. Revision received: 3 August 2001, Accepted: 20 August 2001     

Effects of pinacidil,verapamil, and heart rate on afterdepolarizations in the guinea-pig heart in vivo

Summary Recently, ionic current simulation in the Luo-Rudy model has elucidated putative mechanisms of afterdepolarizations under various experimental conditions. The present study was aimed at gaining insight into the differential mechanism of different types of afterdepolarizations in the guinea-pig heart in vivo. The effects of pharmacological and heart rate perturbations on early (EADs) and delayed (DADs) afterdepolarizations, induced by either digoxin, CsCl, or BayK 8644 were studied, using mid-myocardial left ventricular monophasic action potential (MAP) recordings. Digoxin insignificantly shortened sinus cycle length (SCL) and CsCl and BayK 8644 differentially prolonged SCL and MAP duration. Digoxin induced phase 3-EADs and DADs and CsCl or BayK 8644 induced phase 2- and phase 3-EADs. Pinacidil shortened MAP duration, suppressed almost all the phase 2-EADs and some of the phase 3-EADs, but not the DADs. In a few cases, DADs were manifested following the abolishment of phase 2-EADs by pinacidil, but this phenomenon did not occur in the presence of hexamethonium. Verapamil prolonged SCL, did not significantly affect phase 2-EADs, but suppressed almost all of the DADs, including those which appeared after pinacidil, and all but one of the phase 3-EADs. The effects of pinacidil and verapamil were independent of the mode of afterdepolarization induction. A pacing-induced heart rate increase, which shortened MAP duration, and vagal stimulation, which prolonged MAP duration, attenuated and enhanced phase 2-EADs, respectively. The amplitude of phase 3-EADs was inversely related to the heart rate. These data, taken together, are consistent with those obtained previously by others in a computer model and recent observations on CsCl-induced EADs in the guinea-pig Purkinje fibers in vitro which have indicated that the mechanism of phase 2-EADs is different from that of DADs and that late phase 3-EADs generated under conditions of Ca²⁺ overload and DADs share similar properties.