Jean Chemin

TREK‐1, a K+ channel involved in polymodal pain perception

The TREK‐1 channel is a temperature‐sensitive, osmosensitive and mechano‐gated K+ channel with a regulation by Gs and Gq coupled receptors. This paper demonstrates that TREK‐1 qualifies as one of the molecular sensors involved in pain perception. TREK‐1 is highly expressed in small sensory neurons, is present in both peptidergic and nonpeptidergic neurons and is extensively colocalized with TRPV1, the capsaicin‐activated nonselective ion channel. Mice with a disrupted TREK‐1 gene are more sensitive to painful heat sensations near the threshold between anoxious warmth and painful heat. This phenotype is associated with the primary sensory neuron, as polymodal C‐fibers were found to be more sensitive to heat in single fiber experiments. Knockout animals are more sensitive to low threshold mechanical stimuli and display an increased thermal and mechanical hyperalgesia in conditions of inflammation. They display a largely decreased pain response induced by osmotic changes particularly in prostaglandin E2‐sensitized animals. TREK‐1 appears as an important ion channel for polymodal pain perception and as an attractive target for the development of new analgesics.

Direct inhibition of T-type calcium channels by the endogenous cannabinoid anandamide

Low‐voltage‐activated or T‐type Ca2+ channels (T‐channels) are widely expressed, especially in the central nervous system where they contribute to pacemaker activities and are involved in the pathogenesis of epilepsy. Proper elucidation of their cellular functions has been hampered by the lack of selective pharmacology as well as the absence of generic endogenous regulations. We report here that both cloned (α1G, α1H and α1I subunits) and native T‐channels are blocked by the endogenous cannabinoid, anandamide. Anandamide, known to exert its physiological effects through cannabinoid receptors, inhibits T‐currents independently from the activation of CB1/CB2 receptors, G‐proteins, phospholipases and protein kinase pathways. Anandamide appears to be the first endogenous ligand acting directly on T‐channels at submicromolar concentrations. Block of anandamide membrane transport by AM404 prevents T‐current inhibition, suggesting that anandamide acts intracellularly. Anandamide preferentially binds and stabilizes T‐channels in the inactivated state and is responsible for a significant decrease of T‐currents associated with neuronal firing activities. Our data demonstrate that anandamide inhibition of T‐channels can regulate neuronal excitability and account for CB receptor‐independent effects of this signaling molecule.

Specific contribution of human T‐type calcium channel isotypes (α1G, α1H and α1I) to neuronal excitability

In several types of neurons, firing is an intrinsic property produced by specific classes of ion channels. Low‐voltage‐activated T‐type calcium channels (T‐channels), which activate with small membrane depolarizations, can generate burst firing and pacemaker activity. Here we have investigated the specific contribution to neuronal excitability of cloned human T‐channel subunits. Using HEK‐293 cells transiently transfected with the human α1G (CaV3.1), α1H (CaV3.2) and α1I (CaV3.3) subunits, we describe significant differences among these isotypes in their biophysical properties, which are highlighted in action potential clamp studies. Firing activities occurring in cerebellar Purkinje neurons and in thalamocortical relay neurons used as voltage clamp waveforms revealed that α1G channels and, to a lesser extent, α1H channels produced large and transient currents, while currents related to α1I channels exhibited facilitation and produced a sustained calcium entry associated with the depolarizing after‐potential interval. Using simulations of reticular and relay thalamic neuron activities, we show that α1I currents contributed to sustained electrical activities, while α1G and α1H currents generated short burst firing. Modelling experiments with the NEURON model further revealed that the α1G channel and α1I channel parameters best accounted for T‐channel activities described in thalamocortical relay neurons and in reticular neurons, respectively. Altogether, the data provide evidence for a role of α1I channel in pacemaker activity and further demonstrate that each T‐channel pore‐forming subunit displays specific gating properties that account for its unique contribution to neuronal firing.

Mechanisms underlying excitatory effects of group I metabotropic glutamate receptors via inhibition of 2P domain K+ channels

Group I metabotropic glutamate receptors (mGluRs) are implicated in diverse processes such as learning, memory, epilepsy, pain and neuronal death. By inhibiting background K+ channels, group I mGluRs mediate slow and long‐lasting excitation. The main neuronal representatives of this K+ channel family (K2P or KCNK) are TASK and TREK. Here, we show that in cerebellar granule cells and in heterologous expression systems, activation of group I mGluRs inhibits TASK and TREK channels. D‐myo‐inositol‐1,4,5‐triphosphate and phosphatidyl‐4,5‐inositol‐biphosphate depletion are involved in TASK channel inhibition, whereas diacylglycerols and phosphatidic acids directly inhibit TREK channels. Mechanisms described here with group I mGluRs will also probably stand for many other receptors of hormones and neurotransmitters.

A phospholipid sensor controls mechanogating of the K + channel TREK‐1

TREK‐1 (KCNK2 or K2P2.1) is a mechanosensitive K2P channel that is opened by membrane stretch as well as cell swelling. Here, we demonstrate that membrane phospholipids, including PIP2, control channel gating and transform TREK‐1 into a leak K+ conductance. A carboxy‐terminal positively charged cluster is the phospholipid‐sensing domain that interacts with the plasma membrane. This region also encompasses the proton sensor E306 that is required for activation of TREK‐1 by cytosolic acidosis. Protonation of E306 drastically tightens channel–phospholipid interaction and leads to TREK‐1 opening at atmospheric pressure. The TREK‐1–phospholipid interaction is critical for channel mechano‐, pHi‐ and voltage‐dependent gating.

Alternatively Spliced α1G (CaV3.1) Intracellular Loops Promote Specific T-Type Ca2+ Channel Gating Properties

Abstract At least three genes encode T-type calcium channel α 1 subunits, and identification of cDNA transcripts provided evidence that molecular diversity of these channels can be further enhanced by alternative splicing mechanisms, especially for the α 1G subunit (Ca V 3.1). Using whole-cell patch-clamp procedures, we have investigated the electrophysiological properties of five isoforms of the human α 1G subunit that display a distinct III-IV linker, namely, α 1G-a , α 1G-b , and α 1G-bc , as well as a distinct II-III linker, namely, α 1G-ae , α 1G-be , as expressed in HEK-293 cells. We report that insertion e within the II-III linker specifically modulates inactivation, steady-state kinetics, and modestly recovery from inactivation, whereas alternative splicing within the III-IV linker affects preferentially kinetics and voltage dependence of activation, as well as deactivation and inactivation. By using voltage-clamp protocols mimicking neuronal activities, such as cerebellar train of action potentials and thalamic low-threshold spike, we describe that inactivation properties of α 1G-a and α 1G-ae isoforms can support channel behaviors reminiscent to those described in native neurons. Altogether, these data demonstrate that expression of distinct variants for the T-type α 1G subunit can account for specific low-voltage-activated currents observed in neuronal tissues.

Neuronal T-type α1H Calcium Channels Induce Neuritogenesis and Expression of High-Voltage-Activated Calcium Channels in the NG108–15 Cell Line

Neuronal differentiation involves both morphological and electrophysiological changes, which depend on calcium influx. Voltage-gated calcium channels (VGCCs) represent a major route for calcium entry into neurons. The recently cloned low-voltage-activated T-type calcium channels (T-channels) are the first class of VGCCs functionally expressed in most developing neurons, as well as in neuroblastoma cell lines, but their roles in neuronal development are yet unknown. Here, we document the part played by T-channels in neuronal differentiation. Using NG108–15, a cell line that recapitulates early steps of neuronal differentiation, we demonstrate that blocking T-currents by nickel, mibefradil, or the endogenous cannabinoid anandamide prevents neuritogenesis without affecting neurite outgrowth. Similar results were obtained using antisense oligodeoxynucleotides directed against the α1H T-channel subunit. Furthermore, we describe that inhibition of α1H T-channel activity impairs concomitantly, but independently, both high-voltage-activated calcium channel expression and neuritogenesis, providing strong evidence for a dual role of T-channels in both morphological and electrical changes at early stages of neuronal differentiation.

Cross-talk between the mechano-gated K2P channel TREK-1 and the actin cytoskeleton.

TREK‐1 (KCNK2) is a K2P channel that is highly expressed in fetal neurons. This K+ channel is opened by a variety of stimuli, including membrane stretch and cellular lipids. Here, we show that the expression of TREK‐1 markedly alters the cytoskeletal network and induces the formation of actin‐ and ezrin‐rich membrane protrusions. The genetic inactivation of TREK‐1 significantly alters the growth cone morphology of cultured embryonic striatal neurons. Cytoskeleton remodelling is crucially dependent on the protein kinase A phosphorylation site S333 and the interactive proton sensor E306, but is independent of channel permeation. Conversely, the actin cytoskeleton tonically represses TREK‐1 mechano‐sensitivity. Thus, the dialogue between TREK‐1 and the actin cytoskeleton might influence both synaptogenesis and neuronal electrogenesis.

Overexpression of T‐type calcium channels in HEK‐293 cells increases intracellular calcium without affecting cellular proliferation

Increased expression of low voltage‐activated, T‐type Ca2+ channels has been correlated with a variety of cellular events including cell proliferation and cell cycle kinetics. The recent cloning of three genes encoding T‐type α1 subunits, α1G, α1H and α1I, now allows direct assessment of their involvement in mediating cellular proliferation. By overexpressing the human α1G and α1H subunits in human embryonic kidney (HEK‐293) cells, we describe here that, although T‐type channels mediate increases in intracellular Ca2+ concentrations, there is no significant change in bromodeoxyuridine incorporation and flow cytometric analysis. These results demonstrate that expressions of T‐type Ca2+ channels are not sufficient to modulate cellular proliferation of HEK‐293 cells.

Subunit‐specific modulation of T‐type calcium channels by zinc

Zinc (Zn2+) functions as a signalling molecule in the nervous system and modulates many ionic channels. In this study, we have explored the effects of Zn2+ on recombinant T‐type calcium channels (CaV3.1, CaV3.2 and CaV3.3). Using tsA‐201 cells, we demonstrate that CaV3.2 current (IC50, 0.8 μm) is significantly more sensitive to Zn2+ than are CaV3.1 and CaV3.3 currents (IC50, 80 μm and ∼160 μm, respectively). This inhibition of CaV3 currents is associated with a shift to more negative membrane potentials of both steady‐state inactivation for CaV3.1, CaV3.2 and CaV3.3 and steady‐state activation for CaV3.1 and CaV3.3 currents. We also document changes in kinetics, especially a significant slowing of the inactivation kinetics for CaV3.1 and CaV3.3, but not for CaV3.2 currents. Notably, deactivation kinetics are significantly slowed for CaV3.3 current (∼100‐fold), but not for CaV3.1 and CaV3.2 currents. Consequently, application of Zn2+ results in a significant increase in CaV3.3 current in action potential clamp experiments, while CaV3.1 and CaV3.2 currents are significantly reduced. In neuroblastoma NG 108‐15 cells, the duration of CaV3.3‐mediated action potentials is increased upon Zn2+ application, indicating further that Zn2+ behaves as a CaV3.3 channel opener. These results demonstrate that Zn2+ exhibits differential modulatory effects on T‐type calcium channels, which may partly explain the complex features of Zn2+ modulation of the neuronal excitability in normal and disease states.