Maternal treatment with a selective delta-opioid receptor agonist during gestation has a sex-specific pro-cognitive action in offspring: mechanisms involved.

Background: There is growing evidence that the treatment of several mental disorders can potentially benefit from activation of delta-opioid receptors. In the future, delta-agonists with a safe pharmacological profile can be used for the treatment of mood disorders in pregnant women. However, the data on prenatal exposure to delta-opioid agonists are missing. The present study is aimed to test the hypothesis that the activation of delta-opioid receptors during gravidity has positive effects on the behaviour accompanied by changes in glutamate and monoamine neurotransmission. Methods: Gestating Wistar rats were chronically treated with a selective delta-agonist SNC80 or vehicle. Adult male and female offspring underwent novel object recognition (for the assessment of cognition) and open field (for the assessment of anxiety and habituation) tests, followed by in vivo electrophysiological examination of the activity of hippocampal glutamate and midbrain serotonin (5-HT) and dopamine neurons. Results: We found that the maternal treatment with SNC80 did not affect the offspring's anxiety, habituation, and 5-HT neuronal firing activity. Female offspring of SNC80-treated dams exhibited improved novelty recognition associated with decreased firing rate and burst activity of glutamate and dopamine neurons. Conclusion: Maternal treatment with delta-opioid agonists during gestation may have a pro-cognitive effect on offspring without any negative effects on anxiety and habituation. The putative pro-cognitive effect might be mediated via mechanism(s) involving the firing activity of hippocampal glutamate and mesolimbic dopamine neurons.

Effects of pre-gestational exposure to the stressors and perinatal bupropion administration on the firing activity of serotonergic neurons and anxiety-like behavior in rats.

Exposure by women to stressors before pregnancy increases their risk of contracting prenatal depression, a condition which typically may require antidepressant treatment. And even though such perinatal antidepressant treatment is generally considered to be safe. For the mother, its effects on the development and functioning of the offspring`s brain remain unknown. In this study, we aimed to investigate the effects of pregestational chronic unpredictable stress (CUS) and perinatal bupropion on the anxiety behavior and firing activity of the dorsal raphe nucleus (DRN) serotonin (5-HT) neurons. Female rats underwent CUS for three weeks before mating. Bupropion was administered to them from gestation day ten until their offspring were weaned. Behavioral (elevated plus maze or EPM test) and neurophysiological (single-unit in vivo electrophysiology) assessments were performed on offspring who reached the age of 48-56 days. We found that maternal CUS and perinatal bupropion, as separate factors on their own, did not change offspring behavior. There was, however, an interaction between their effects on the number of entries to the open arms and time spent in the intersection: maternal CUS tended to decrease these values, and perinatal bupropion tended to diminish CUS effect. Maternal CUS increased the firing activity of 5-HT neurons in males, but not females. Perinatal bupropion did not alter the firing activity of 5-HT neurons but tended to potentiate the maternal CUS-induced increase in 5-HT neuronal firing activity. The CUS-induced increase in firing activity of 5-HT neurons might be a compensatory mechanism that diminishes the negative effects of maternal stress. Perinatal bupropion does not alter the offspring`s anxiety and firing activity of 5-HT, but it does intervene in the effects of maternal stress.

Effects of the acute administration of delta-opioid receptor ligands on the excitability of rat hippocampal glutamate and brainstem monoamine neurons in vivo.

It was previously reported that the delta opioid receptor (DOR) agonist SNC80 and antagonist naltrindole modulate the excitability of hippocampal glutamate neurons in primary cultures. The present study aimed to investigate the acute effects of these ligands on the firing activity of hippocampal cornu ammonis 1/3 (CA1/3) glutamate, dorsal raphe nucleus (DRN) serotonin (5-HT), locus coeruleus (LC) noradrenaline, and ventral tegmental area (VTA) dopamine neurons in in vivo conditions. Adult Wistar male rats were used. SNC80 and naltrindole were administered intravenously. Neuronal firing activity was assessed using extracellular single-unit electrophysiology. SNC80, administered first at 1-3 mg/kg, dose-dependently inhibited CA1/3 glutamate, DRN 5-HT, and VTA dopamine neurons. Naltrindole, administered at 1-3 mg/kg after SNC80, did not have any additional effect. Naltrindole, administered first at 1-3 mg/kg, stimulated DRN 5-HT neurons in a dose-dependent manner; this stimulation was dose-dependently reversed by 1-3 mg/kg of SNC80. SNC80 and naltrindole inhibited LC noradrenaline neurons when only they were co-administered at 3 mg/kg, and only when SNC80 was administered first. In conclusion, DOR ligands alter the firing activity of hippocampal glutamate and brainstem monoamine neurons in in vivo conditions. The psychoactive effects of DOR ligands, reported in previous studies, might be explained, at least in part, by their ability to modulate the firing activity of hippocampal glutamate and brainstem monoamine neurons.

Effects of chronic delta-opioid receptor agonist on the excitability of hippocampal glutamate and brainstem monoamine neurons, anxiety, locomotion, and habituation in rats.

BACKGROUND: Short-term treatment with non-peptide agonists of delta-opioid receptors, such as agonist SNC80, induced behavioral effects in rodents, which could be modulated via changes in central neurotransmission. The present experiments aimed at testing the hypothesis that chronic treatment with SNC80 induces anxiolytic effects associated with changes in hippocampal glutamate and brainstem monoamine pathways. METHODS: Adult male Wistar rats were used in experiments. Rats were treated with SNC80 (3 mg/kg/day) for fourteen days. Neuronal excitability was assessed using extracellular in vivo single-unit electrophysiology. The behavioral parameters were examined using the elevated plus maze and open field tests. RESULTS: Chronic SNC80 treatment increased the excitability of hippocampal glutamate and ventral tegmental area dopamine neurons and had no effect on the firing activity of dorsal raphe nucleus serotonin cells. Chronic SNC80 treatment induced anxiolytic effects, which were, however, confounded by increased locomotor activity clearly confirmed in an open field test. The ability to cope with stressful situations and habituation processes in a novel environment was not influenced by chronic treatment with SNC80. CONCLUSION: Our study suggests that the psychoactive effects of SNC80 might be explained by its ability to stimulate hippocampal glutamate and mesolimbic dopamine transmission.

Stress, depression, and hippocampus: from biochemistry to electrophysiology.

Major depressive disorder is a very common serious mental illness with increasing prevalence in the population. Its pathology includes biochemical, morphological, and electrophysiological changes in various brain areas. In spite of decades of extensive research pathophysiology of depression is still not sufficiently understood. When depression occurs just before or during pregnancy, it may have a detrimental effect on perinatal and/or postnatal brain development, affecting the offspring's behavior. An important role in the pathology of depression is the hippocampus as a center for cognition and memory. Here we review changes in morphology, biochemical, and electrical signaling caused by depression in first and second generation identified in various animal models.

Clozapine Reverses Dysfunction of Glutamatergic Neurons Derived From Clozapine-Responsive Schizophrenia Patients.

The cellular pathology of schizophrenia and the potential of antipsychotics to target underlying neuronal dysfunctions are still largely unknown. We employed glutamatergic neurons derived from induced pluripotent stem cells (iPSC) obtained from schizophrenia patients with known histories of response to clozapine and healthy controls to decipher the mechanisms of action of clozapine, spanning from molecular (transcriptomic profiling) and cellular (electrophysiology) levels to observed clinical effects in living patients. Glutamatergic neurons derived from schizophrenia patients exhibited deficits in intrinsic electrophysiological properties, synaptic function and network activity. Deficits in K+ and Na+ currents, network behavior, and glutamatergic synaptic signaling were restored by clozapine treatment, but only in neurons from clozapine-responsive patients. Moreover, neurons from clozapine-responsive patients exhibited a reciprocal dysregulation of gene expression, particularly related to glutamatergic and downstream signaling, which was reversed by clozapine treatment. Only neurons from clozapine responders showed return to normal function and transcriptomic profile. Our results underscore the importance of K+ and Na+ channels and glutamatergic synaptic signaling in the pathogenesis of schizophrenia and demonstrate that clozapine might act by normalizing perturbances in this signaling pathway. To our knowledge this is the first study to demonstrate that schizophrenia iPSC-derived neurons exhibit a response phenotype correlated with clinical response to an antipsychotic. This opens a new avenue in the search for an effective treatment agent tailored to the needs of individual patients.

Voltage-dependent CaV3.2 and CaV2.2 channels in nociceptive pathways.

Noxious stimuli like cold, heat, pH change, tissue damage, and inflammation depolarize a membrane of peripheral endings of specialized nociceptive neurons which eventually results in the generation of an action potential. The electrical signal is carried along a long axon of nociceptive neurons from peripheral organs to soma located in dorsal root ganglions and further to the dorsal horn of the spinal cord where it is transmitted through a chemical synapse and is carried through the spinal thalamic tract into the brain. Two subtypes of voltage-activated calcium play a major role in signal transmission: a low voltage-activated CaV3.2 channel and a high voltage-activated CaV2.2 channel. The CaV3.2 channel contributes mainly to the signal conductance along nociceptive neurons while the principal role of the CaV2.2 channel is in the synaptic transmission at the dorsal horn. Both channels contribute to the signal initiation at peripheral nerve endings. This review summarizes current knowledge about the expression and distribution of these channels in a nociceptive pathway, the regulation of their expression and gating during pain pathology, and their suitability as targets for pharmacological therapy.

Secretory carrier-associated membrane protein 2 (SCAMP2) regulates cell surface expression of T-type calcium channels.

Low-voltage-activated T-type Ca2+ channels are key regulators of neuronal excitability both in the central and peripheral nervous systems. Therefore, their recruitment at the plasma membrane is critical in determining firing activity patterns of nerve cells. In this study, we report the importance of secretory carrier-associated membrane proteins (SCAMPs) in the trafficking regulation of T-type channels. We identified SCAMP2 as a novel Cav3.2-interacting protein. In addition, we show that co-expression of SCAMP2 in mammalian cells expressing recombinant Cav3.2 channels caused an almost complete drop of the whole cell T-type current, an effect partly reversed by single amino acid mutations within the conserved cytoplasmic E peptide of SCAMP2. SCAMP2-induced downregulation of T-type currents was also observed in cells expressing Cav3.1 and Cav3.3 channel isoforms. Finally, we show that SCAMP2-mediated knockdown of the T-type conductance is caused by the lack of Cav3.2 expression at the cell surface as evidenced by the concomitant loss of intramembrane charge movement without decrease of total Cav3.2 protein level. Taken together, our results indicate that SCAMP2 plays an important role in the trafficking of Cav3.2 channels at the plasma membrane.

De novo SCN8A and inherited rare CACNA1H variants associated with severe developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are a group of severe epilepsies that are characterized by seizures and developmental delay. DEEs are primarily attributed to genetic causes and an increasing number of cases have been correlated with variants in ion channel genes. In this study, we report a child with an early severe DEE. Whole exome sequencing showed a de novo heterozygous variant (c.4873-4881 duplication) in the SCN8A gene and an inherited heterozygous variant (c.952G > A) in the CACNA1H gene encoding for Nav1.6 voltage-gated sodium and Cav3.2 voltage-gated calcium channels, respectively. In vitro functional analysis of human Nav1.6 and Cav3.2 channel variants revealed mild but significant alterations of their gating properties that were in general consistent with a gain- and loss-of-channel function, respectively. Although additional studies will be required to confirm the actual pathogenic involvement of SCN8A and CACNA1H, these findings add to the notion that rare ion channel variants may contribute to the etiology of DEEs.

Di-4-ANEPPS Modulates Electrical Activity and Progress of Myocardial Ischemia in Rabbit Isolated Heart.

AIMS: Although voltage-sensitive dye di-4-ANEPPS is a common tool for mapping cardiac electrical activity, reported effects on electrophysiological parameters are rather. The main goals of the study were to reveal effects of the dye on rabbit isolated heart and to verify, whether rabbit isolated heart stained with di-4-ANEPPS is a suitable tool for myocardial ischemia investigation. METHODS AND RESULTS: Study involved experiments on stained (n = 9) and non-stained (n = 11) Langendorff perfused rabbit isolated hearts. Electrophysiological effects of the dye were evaluated by analysis of various electrogram (EG) parameters using common paired and unpaired statistical tests. It was shown that staining the hearts with di-4-ANEPPS leads to only short-term sporadic prolongation of impulse conduction through atria and atrioventricular node. On the other hand, significant irreversible slowing of heart rate and ventricular conduction were found in stained hearts as compared to controls. In patch clamp experiments, significant inhibition of sodium current density was observed in differentiated NG108-15 cells stained by the dye. Although no significant differences in mean number of ventricular premature beats were found between the stained and the non-stained hearts in ischemia as well as in reperfusion, all abovementioned results indicate increased arrhythmogenicity. In isolated hearts during ischemia, prominent ischemic patterns appeared in the stained hearts with 3-4 min delay as compared to the non-stained ones. Moreover, the ischemic changes did not achieve the same magnitude as in controls even after 10 min of ischemia. It resulted in poor performance of ischemia detection by proposed EG parameters, as was quantified by receiver operating characteristics analysis. CONCLUSION: Our results demonstrate significant direct irreversible effect of di-4-ANEPPS on spontaneous heart rate and ventricular impulse conduction in rabbit isolated heart model. Particularly, this should be considered when di-4-ANEPPS is used in ischemia studies in rabbit. Delayed attenuated response of such hearts to ischemia might lead to misinterpretation of obtained results.

Maternal immune activation in rats attenuates the excitability of monoamine-secreting neurons in adult offspring in a sex-specific way.

Higher risk of depression and schizophrenia in descendants of mothers experienced acute infection during the pregnancy has been reported. Since monoamines are fundamental in mentioned psychopathologies, it is possible that maternal immune activation leads to impaired functioning of serotonin (5-HT), noradrenaline, and dopamine neurons in offspring. To test this hypothesis, we examined the effect of maternal immune activation by lipopolysaccharide (LPS) in rats on the excitability of monoamine-secreting neurons in the offspring. LPS was administered during days 15-19 of the gestation in the rising doses of 20-80 µg/kg; control dams received vehicle. During days 53-63 postpartum, rats were anesthetized and electrodes were inserted into the dorsal raphe nucleus, locus coeruleus, and ventral tegmental area for in vivo excitability assessment of 5-HT, noradrenaline, and dopamine neurons. Maternal immune activation suppressed the firing rate of 5-HT neurons in both sexes and stimulated the firing rate of dopamine neurons in males. Decrease in the firing rate of 5-HT neurons was accompanied with an increase, and increase in the firing rate of dopamine neurons with a decrease, in the density of spontaneously active cells. Maternal immune activation also decreased the variability of interspike intervals in 5-HT and dopamine neurons. It is possible that the alteration of excitability of 5-HT and dopamine neurons by maternal immune activation is involved in the psychopathologies induced by infectious disease during the pregnancy. Stimulation of dopamine excitability in males might be a compensatory mechanism secondary to the maternal immune challenge-induced suppression of 5-HT neurons.

Delta-opioid receptor-mediated modulation of excitability of individual hippocampal neurons: mechanisms involved.

BACKGROUND: Delta-opioid receptor (DOR)-mediated modulation of hippocampal neural networks is involved in emotions, cognition, and in pathophysiology and treatment of mood disorders. In this study, we examined the effects of DOR agonist (SNC80) and antagonist (naltrindole) on the excitability of individual hippocampal neurons. METHODS: Primary neuronal cultures were prepared from hippocampi of newborn rats and cultivated in vitro for 8-14 days (DIV8-14). The effects of SNC80 naltrindole on evoked and spontaneous action potentials (APs) were measured at DIV8-9 and DIV13-14, respectively. RESULTS: SNC80 (100 µM) potentiated spontaneous AP firing and stimulated sodium current; naltrindole had opposite effects. The stimulatory effect of 100 µM of SNC80 was revoked by pre-administration of 1 µM of naltrindole. SNC80 and naltrindole induced similar inhibitory effects on the evoked AP firing and on the calcium current. Further, SNC80 inhibited both peak and sustained potassium currents. Naltrindole had no effect on potassium currents. CONCLUSION: We suggest that the effects of naltrindole and high concentration of SNC80 on the sodium currents are mediated via DORs and underlying the changes in spontaneous activity. The inhibitory effects of SNC80 on calcium and potassium currents might also be DOR-dependent; these currents might mediate SNC80 effect on the evoked AP firing. The inhibitory effects of naltrindole on calcium and of low doses of SNC80 on sodium currents might be however DOR independent. The behavioral effects of SNC80 and naltrindole, observed in previous studies, might be mediated, at least in part, via the modulatory effect of these ligands on the excitability of hippocampal neurons.

Modulation of voltage-gated CaV2.2 Ca2+ channels by newly identified interaction partners.

Voltage-gated Ca2+ channels are typically integrated in a complex network of protein-protein-interactions, also referred to as Ca2+ channel nanodomains. Amongst the neuronal CaV2 channel family, CaV2.2 is of particular importance due to its general role for signal transmission from the periphery to the central nervous system, but also due to its significance for pain perception. Thus, CaV2.2 is an ideal target candidate to search for pharmacological inhibitors but also for novel modulatory interactors. In this review we summarize the last years findings of our intense screenings and characterization of the six CaV2.2 interaction partners, tetraspanin-13 (TSPAN-13), reticulon 1 (RTN1), member 1 of solute carrier family 38 (SLC38), prostaglandin D2 synthase (PTGDS), transmembrane protein 223 (TMEM223), and transmembrane BAX inhibitor motif 3 (Grina/TMBIM3) containing protein. Each protein shows a unique way of channel modulation as shown by extensive electrophysiological studies. Amongst the newly identified interactors, Grina/TMBIM3 is most striking due to its modulatory effect which is rather comparable to G-protein regulation.

A Study among the Genotype, Functional Alternations, and Phenotype of 9 SCN1A Mutations in Epilepsy Patients.

Mutations in the voltage-gated sodium channel Nav1.1 (SCN1A) are linked to various epileptic phenotypes with different severities, however, the consequences of newly identified SCN1A variants on patient phenotype is uncertain so far. The functional impact of nine SCN1A variants, including five novel variants identified in this study, was studied using whole-cell patch-clamp recordings measurement of mutant Nav1.1 channels expressed in HEK293T mammalian cells. E78X, W384X, E1587K, and R1596C channels failed to produce measurable sodium currents, indicating complete loss of channel function. E788K and M909K variants resulted in partial loss of function by exhibiting reduced current density, depolarizing shifts of the activation and hyperpolarizing shifts of the inactivation curves, and slower recovery from inactivation. Hyperpolarizing shifts of the activation and inactivation curves were observed in D249E channels along with slower recovery from inactivation. Slower recovery from inactivation was observed in E78D and T1934I with reduced current density in T1934I channels. Various functional effects were observed with the lack of sodium current being mainly associated with severe phenotypes and milder symptoms with less damaging channel alteration. In vitro functional analysis is thus fundamental for elucidation of the molecular mechanisms of epilepsy, to guide patients' treatment, and finally indicate misdiagnosis of SCN1A related epilepsies.

A rare CACNA1H variant associated with amyotrophic lateral sclerosis causes complete loss of Cav3.2 T-type channel activity.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of cortical, brain stem and spinal motor neurons that leads to muscle weakness and death. A previous study implicated CACNA1H encoding for Cav3.2 calcium channels as a susceptibility gene in ALS. In the present study, two heterozygous CACNA1H variants were identified by whole genome sequencing in a small cohort of ALS patients. These variants were functionally characterized using patch clamp electrophysiology, biochemistry assays, and molecular modeling. A previously unreported c.454GTAC > G variant produced an inframe deletion of a highly conserved isoleucine residue in Cav3.2 (p.ΔI153) and caused a complete loss-of-function of the channel, with an additional dominant-negative effect on the wild-type channel when expressed in trans. In contrast, the c.3629C > T variant caused a missense substitution of a proline with a leucine (p.P1210L) and produced a comparatively mild alteration of Cav3.2 channel activity. The newly identified ΔI153 variant is the first to be reported to cause a complete loss of Cav3.2 channel function. These findings add to the notion that loss-of-function of Cav3.2 channels associated with rare CACNA1H variants may be risk factors in the complex etiology of ALS.

Role of glucocorticoid- and monoamine-metabolizing enzymes in stress-related psychopathological processes.

Glucocorticoid signaling is fundamental in healthy stress coping and in the pathophysiology of stress-related diseases, such as post-traumatic stress disorder (PTSD). Glucocorticoids are metabolized by cytochrome P450 (CYP) as well as 11-ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) and 2 (11ßHSD2). Acute stress-induced increase in glucocorticoid concentrations stimulates the expression of several CYP sub-types. CYP is primarily responsible for glucocorticoid metabolism and its increased activity can result in decreased circulating glucocorticoids in response to repeated stress stimuli. In addition, repeated stress-induced glucocorticoid release can promote 11ßHSD1 activation and 11ßHSD2 inhibition, and the 11ßHSD2 suppression can lead to apparent mineralocorticoid excess. The activation of CYP and 11ßHSD1 and the suppression of 11ßHSD2 may at least partly contribute to development of the blunted glucocorticoid response to stressors characteristic in high trait anxiety, PTSD, and other stress-related disorders. Glucocorticoids and glucocorticoid-metabolizing enzymes interact closely with other biomolecules such as inflammatory cytokines, monoamines, and some monoamine-metabolizing enzymes, namely the monoamine oxidase type A (MAO-A) and B (MAO-B). Glucocorticoids boost MAO activity and this decreases monoamine levels and induces oxidative tissue damage which then activates inflammatory cytokines. The inflammatory cytokines suppress CYP expression and activity. This dynamic cross-talk between glucocorticoids, monoamines, and their metabolizing enzymes could be a critical factor in the pathophysiology of stress-related disorders.Lay summaryGlucocorticoids, which are produced and released under the control by brain regulatory centers, are fundamental in the stress response. This review emphasizes the importance of glucocorticoid metabolism and particularly the interaction between the brain and the liver as the major metabolic organ in the body. The activity of enzymes involved in glucocorticoid metabolism is proposed to play not only an important role in positive, healthy glucocorticoid effects, but also to contribute to the development and course of stress-related diseases.

Pre-gestational stress impacts excitability of hippocampal cells in vitro and is associated with neurobehavioral alterations during adulthood.

Chronic stress during pregnancy or even prior to gestation can negatively affect offspring´s neurobehavioural development. Several studies have shown, that offspring who had experienced excessive stress during gestation had higher rates of cognitive and mood disorders later during adolescence or in adulthood. Hippocampal neurons play a crucial role in the regulation of behavior, mainly in anxiety-related behaviors and spatial learning and memory. Recently, it has been shown, that excessive stress even prior to gestation could interfere with sensitive developmental processes in the brain and may affect hippocampal functioning with severe neurobehavioural consequences in later life. The aim of this work was to investigate the effects of pre-gestational stress of the rat dams on the hippocampal excitability of the pups right after the birth. Neurobehavioural consequences of pre-gestational stress were analyzed during adolescence (35-40 postnatal days) and in early adulthood (75-80 postnatal days). We have shown that even pre-gestational chronic maternal stress increased resting membrane potential, suppressed depolarization-activated action potential firing, and increased spontaneous activity of hippocampal cells from newborn offspring. Altered function of hippocampus was reflected at the behavioural level. Adolescent male offspring of dams exposed stress prior to conception showed hyperactivity-like behaviour in a new stressful environment and increased anxiety-like behaviour during adulthood compared to adult males from non-stress group. Together, this work suggests, that chronic stress even prior to gestation can interfere with functional brain development of the offspring and can cause long-term behavioural changes at the level of neurobehavioural adaptations.

Grina/TMBIM3 modulates voltage-gated CaV2.2 Ca2+ channels in a G-protein-like manner.

Grina/TMBIM3 is a poorly characterized transmembrane protein with a broad expression pattern in mammals and with a very ancient origin within eukaryotes. Although initially characterized as an NMDA-receptor associated subunit, there is increasing evidence that Grina/TMBIM3 is involved in the unfolded protein response and controls apoptosis via regulation of Ca2+ homeostasis. Here, we investigate a putative direct interaction of Grina/TMBIM3 with voltage gated Ca2+ channels, in particular with the CaV2.2 α1-subunit and describe its modulatory effects on the current through CaV2.2 N-type channels. Direct interaction was confirmed by co-immunoprecipitation studies and membrane localization was proven. Co-expression of Grina/TMBIM3 with CaV2.2 channels resulted in a significant decrease of the current amplitude and in a slowing of the kinetics of current activation. This effect was accompanied by a significant shift of the voltage dependencies of activation time constants towards more depolarized voltages. Application of a stimulus protocol including a strong depolarizing pulse relieved inhibition of current amplitude by Grina/TMBIM3. When Grina/TMBIM3 was present, inactivation by an action potential-like train of pulses was diminished. Both observations resemble mechanisms that are well-studied modulatory effects of G-protein ßγ subunits on CaV2 channels. The impact of Grina/TMBIM3 and G-protein ßγ subunits are rather comparable with respect to suppression of current amplitude and slowing of activation kinetics. Furthermore, both modulators had the same effect on current inactivation when evoked by an action potential-like train of pulses.

Identification of a molecular gating determinant within the carboxy terminal region of Cav3.3 T-type channels.

The physiological functions controlled by T-type channels are intrinsically dependent on their gating properties, and alteration of T-type channel activity is linked to several human disorders. Therefore, it is essential to develop a clear understanding of the structural determinants responsible for the unique gating features of T-type channels. Here, we have investigated the specific role of the carboxy terminal region by creating a series a deletion constructs expressed in tsA-201 cells and analyzing them by patch clamp electrophysiology. Our data reveal that the proximal region of the carboxy terminus contains a structural determinant essential for shaping several gating aspects of Cav3.3 channels, including voltage-dependence of activation and inactivation, inactivation kinetics, and coupling between the voltage sensing and the pore opening of the channel. Altogether, our data are consistent with a model in which the carboxy terminus stabilizes the channel in a closed state.