The Effect of Calcium Ions on the Electrophysiological Properties of Single ANO6 Channels.

Proteins belonging to the anoctamin (ANO) family form calcium-activated chloride channels (CaCCs). The most unusual member of this family, ANO6 (TMEM16F), simultaneously exhibits the functions of calcium-dependent scramblase and the ion channel. ANO6 affects the plasma membrane dynamics and phosphatidylserine transport; it is also involved in programmed cell death. The properties of ANO6 channels remain the subject of debate. In this study, we investigated the effect of variations in the intracellular and extracellular concentrations of calcium ions on the electrophysiological properties of endogenous ANO6 channels by recording single ANO6 channels. It has been demonstrated that (1) a high calcium concentration in an extracellular solution increases the activity of endogenous ANO6 channels, (2) the permeability of endogenous ANO6 channels for chloride ions is independent of the extracellular concentration of calcium ions, (3) that an increase in the intracellular calcium concentration leads to the activation of endogenous ANO6 channels with double amplitude, and (4) that the kinetics of the channel depend on the plasma membrane potential rather than the intracellular concentration of calcium ions. Our findings give grounds for proposing new mechanisms for the regulation of the ANO6 channel activity by calcium ions both at the inner and outer sides of the membrane.

Calcium chelation independent effects of BAPTA on endogenous ANO6 channels in HEK293T cells.

Selective calcium chelator 1,2-Bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) is a common tool to investigate calcium signaling. However, BAPTA expresses various effects on intracellular calcium signaling, which are not related to its ability to bind Ca2+. In patch clamp experiments, we investigated calcium chelation independent effects of BAPTA on endogenous calcium-activated chloride channels ANO6 (TMEM16F) in HEK293T cells. We have found that application of BAPTA to intracellular solution led to two distinct effects on channels properties. On the one hand, application of BAPTA acutely reduced amplitude of endogenous ANO6 channels induced by 10 µM Ca2+ in single channel recordings. On the other hand, BAPTA application by itself induced ANO6 channel activity in the absence of the intracellular calcium elevation. Open channel probability was enhanced by increasing the intracellular BAPTA concentration from 0.1 to 1 and 10 mM. Another calcium chelator EGTA did not demonstrate chelation independent effects on the ANO6 activity in the same conditions. Due to off-target effects BAPTA should be used with caution when studying calcium-activated ANO6 channels.

Role of STIM2 and Orai proteins in regulating TRPC1 channel activity upon calcium store depletion.

Store-operated calcium channels are the major player in calcium signaling in non-excitable cells. Store-operated calcium entry is associated with the Orai, stromal interaction molecule (STIM), and transient receptor potential canonical (TRPC) protein families. Researchers have provided conflicting data about TRPC1 channel regulation by Orai and STIM. To determine how Orai and STIM influence endogenous TRPC1 pore properties and regulation, we used single channel patch-clamp recordings. Here we showed that knockout or knockdown of Orai1 or Orai3 or overexpression of the dominant-negative mutant Orai1 E106Q did not change the conductance or selectivity of single TRPC1 channels. In addition, these TRPC1 channel properties did not depend on the amount of STIM1 and STIM2 proteins. To study STIM2-mediated regulation of TRPC1 channels, we utilized partial calcium store depletion induced by application of 10 nM thapsigargin (Tg). TRPC1 activation by endogenous STIM2 was greatly decreased in acute extracellular calcium-free experiments. STIM2 overexpression increased both the basal activity and number of silent TRPC1 channels in the plasma membrane. After calcium store depletion, overexpressed STIM2 directly activated TRPC1 in the plasma membrane even without calcium entry in acute experiments. However, this effect was abrogated by co-expression with the non-permeable Orai1 E106Q mutant protein. Taken together, our single-channel patch clamp experiments clearly demonstrated that endogenous TRPC1 forms a channel pore without involving Orai proteins. Calcium entry through Orai triggered TRPC1 channel activation in the plasma membrane, while subsequent STIM2-mediated TRPC1 activity regulation was not dependent on calcium entry.

A Novel Modulator of STIM2-Dependent Store-Operated Ca2+ Channel Activity.

Store-operated Ca2+ entry is one of the main pathways of calcium influx into non-excitable cells, which entails the initiation of many intracellular processes. The endoplasmic reticulum Ca2+ sensors STIM1 and STIM2 are the key components of store-operated Ca2+ entry in mammalian cells. Under physiological conditions, STIM proteins are responsible for store-operated Ca2+ entry activation. The STIM1 and STIM2 proteins differ in their potency for activating different store-operated channels. At the moment, there are no selective modulators of the STIM protein activity. We screened a library of small molecules and found the 4-MPTC compound, which selectively inhibited STIM2-dependent store-operated Ca2+ entry (IC50 = 1 µM) and had almost no effect on the STIM1-dependent activation of store-operated channels.

Molecular Pathogenesis in Huntington's Disease.

Huntington's disease (HD) is a severe autosomal dominant neurodegenerative disorder characterized by a combination of motor, cognitive, and psychiatric symptoms, atrophy of the basal ganglia and the cerebral cortex, and inevitably progressive course resulting in death 5-20 years after manifestation of its symptoms. HD is caused by expansion of CAG repeats in the HTT gene, which leads to pathological elongation of the polyglutamine tract within the respective protein - huntingtin. In this review, we present a modern view on molecular biology of HD as a representative of the group of polyglutamine diseases, with an emphasis on conformational changes of mutant huntingtin, disturbances in its cellular processing, and proteolytic stress in degenerating neurons. Main pathogenetic mechanisms of neurodegeneration in HD are discussed in detail, such as systemic failure of transcription, mitochondrial dysfunction and suppression of energy metabolism, abnormalities of cytoskeleton and axonal transport, microglial inflammation, decrease in synthesis of brain-derived neurotrophic factor, etc.

Huntington's Disease: Calcium Dyshomeostasis and Pathology Models.

Huntington's disease (HD) is a severe inherited neurodegenerative disorder characterized by motor dysfunction, cognitive decline, and mental impairment. At the molecular level, HD is caused by a mutation in the first exon of the gene encoding the huntingtin protein. The mutation results in an expanded polyglutamine tract at the N-terminus of the huntingtin protein, causing the neurodegenerative pathology. Calcium dyshomeostasis is believed to be one of the main causes of the disease, which underlies the great interest in the problem among experts in molecular physiology. Recent studies have focused on the development of animal and insect HD models, as well as patient-specific induced pluripotent stem cells (HD-iPSCs), to simulate the disease's progression. Despite a sesquicentennial history of HD studies, the issues of diagnosis and manifestation of the disease have remained topical. The present review addresses these issues.

STIM1 Protein Activates Store-Operated Calcium Channels in Cellular Model of Huntington's Disease.

We have shown that the expression of full-length mutated huntingtin in human neuroblastoma cells (SK-N-SH) leads to an abnormal increase in calcium entry through store-operated channels. In this paper, the expression of the N-terminal fragment of mutated huntingtin (Htt138Q-1exon) is shown to be enough to provide an actual model for Huntington's disease. We have shown that Htt138Q-1exon expression causes increased store-operated calcium entry, which is mediated by at least two types of channels in SK-N-SH cells with different reversal potentials. Calcium sensor, STIM1, is required for activation of store-operated calcium entry in these cells. The results provide grounds for considering the proteins responsible for the activation and maintenance of the store-operated calcium entry as promising targets for developing novel therapeutics for neurodegenerative diseases.

Regulation of store-operated channels by scaffold proteins in a431 cells.

Store-operated channels are major calcium influx pathways in nonexitable cells. Homer scaffold proteins are well known for their role in regulating calcium signaling. Here we report on a detailed single-channel level characterization of native store-operated channels regulated by Homer scaffold proteins in A431 carcinoma cells. By applying the single-channel patch-clamp technique, we found that different types of store-operated calcium channels have different sensitivities to Homer proteins.

Selectivity of ATP-activated GTP-dependent Ca(2+)-permeable channels in rat macrophage plasma membrane.

Outside-out configuration of the patch clamp technique was used to test whether an intracellular application of G protein activator (GTP gamma S) affects ATP-activated Ca(2+)-permeable channels in rat macrophages without any agonist in the bath solution. With 145 mM K+ (pCa 8.0) in the pipette solution, activity of channels permeable to a variety of divalent cations and Na+ was observed and general channel characteristics were found to be identical to those of ATP-activated ones. Absence of extracellular ATP makes it possible to avoid the influence of ATP receptor desensitization and to study the channel selectivity using a number of divalent cations (105 mM) and Na+ (145 mM) as the charge carriers. Permeability sequence estimated by extrapolated reversal potential measurements was: Ca2+:Ba2+:Mn2+:Sr2+: Na+:K+ = 68:30:26:10:3.5:1. Slope conductances (in pS) for permeant ions rank as follows: Ca2+:Sr2+: Na+:Mn2+:Ba2+ = 19:18:14:12:10. Unitary Ca2+ currents display a tendency to saturate with the Ca2+ concentration increase with apparent dissociation constant (Kd) of 10 mM. No block of Na+ permeation by extracellular Ca2+ in millimolar range was found. The data obtained suggest that (i) activation of some G protein is sufficient to gate the channels without the ATP receptor being occupied, (ii) the ATP receptor activation results in the gating of a special channel with the properties that differ markedly from those of the receptor-operated or voltage-gated Ca(2+)-permeable channels on the other cell types.

ATP-activated inward current and calcium-permeable channels in rat macrophage plasma membranes.

1. To study mechanisms of receptor-operated Ca2+ influx in non-excitable cells, membrane currents of rat peritoneal macrophages were recorded using whole-cell cell-attached and outside-out configurations of the patch clamp technique. Under whole-cell recording conditions, ATP applied in micromolar concentrations elicited an inward current response when the bath solution contained Ba2+, Ca2+ or Na+ as the only permeant cations. 2. Increasing the Mg2+ concentration had an inhibitory effect on the ATP-induced inward current indicating that the active form of ATP responsible for the cation entry is ATP4-. The nucleotide potency order was ATP > ATP gamma S > ADP. UTP was completely ineffective (n = 19). The data obtained are consistent with the ATP receptor being of the P2Z type. 3. The macrophage plasma membrane was impermeable to Tris+ during the ATP-induced current at ATP4- concentrations varying from 0.07 to 500 microM. At higher concentrations, ATP produced a large inward steady-state current, which could be attributed to membrane permeabilization. 4. Activity of single channels was recorded when ATP was applied to the external surface of the patch membrane both in cell-attached and outside-out experiments. A specific property of the channels appeared to be the existence of at least four conductance sublevels. With 105 mM Ba2+ as the permeant cation, the conductance sublevels were 3.5, 7, 10 and 15 pS. With 10 mM Ca2+ the sublevel conductances were equal to 4, 9, 13 and 17 pS. 5. The unitary conductance estimated from the whole-cell current noise analysis (3.5-4.5 pS for 105 mM Ba2+) was significantly lower than that obtained from single channel measurements at the main (3rd) current level, but it was very close to the conductance of the minimum (1st) level. 6. Extrapolated reversal potential values estimated from current-voltage curves for predominant conductance levels were equal to +40 and +26 mV for 105 mM Ba2+ and 10 mM Ca2+, respectively. The permeability ratios fell in the sequence: PCa:PBa:PK = 71.:29:1. Thus, ATP-activated channels in the macrophage membrane are rather selective for divalent vs. monovalent cations, with the predominant permeability being for Ca2+.

ATP-operated calcium-permeable channels activated via a guanine nucleotide-dependent mechanism in rat macrophages.

1. To elucidate the possible involvement of a G protein in ATP-evoked Ca(2+)-permeable channel activity, membrane currents of rat peritoneal macrophages were recorded using inside-out and cell-attached configurations of the patch clamp technique. 2. In inside-out experiments with a pipette solution containing 105 mM Ba2+, application of 100 microM GTP or GTP gamma S to the internal surface of the membrane elicited a rise in channel activity. This effect was observed in 49% of the patches investigated (n = 69). The mean value of NPo (N, number of open channels; Po, channel open probability) was equal to 0.49 +/- 0.27 (mean +/- S.E.M.; n = 16). The delay in the activity development was 21 +/- 8 s (n = 18) with 200 microM ATP added to the pipette solution and about 4 min (n = 5) without agonist in the pipette. Similar results were obtained with 10 mM Ca2+ as the only permeant cation. 3. Properties of GTP gamma S-evoked channels were identical to those of channels activated by extracellular application of ATP. The channels exhibited at least four conductance sublevels, the 4th one being the least frequent. With 105 mM Ba2+ as a permeant cation, sublevel conductances were 3.5, 7, 10 and 15 pS. Corresponding values for 10 mM Ca2+ were about 4, 9, 13 and 17 pS. Extrapolated reversal potential (Er) values were about +40 and +25 mV for Ba2+ and Ca2+, respectively. 4. The activity of channels with similar characteristics could be induced by the extracellular application of fluoride in cell-attached experiments without any agonist in the pipette solution.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-activated Ca(2+)-permeable channels in rat peritoneal macrophages.

The patch-clamp technique was used to study mechanisms of ATP-induced Ca2+ influx in rat peritoneal macrophages. The experiments on whole-cell and patch membranes have shown that extracellular ATP activates channels permeable to di- and monovalent inorganic cations. Ratios of unitary channel conductances in 105 mM Ca2+, Sr2+, Mn2+, Ba2+ and normal sodium solutions were 1.0, 0.95, 0.75, 0.55 and 0.85, respectively. The channels could open in the presence of non-hydrolyzable GTP analogues in artificial intracellular solution. The data are consistent with the hypothesis that a GTP-binding protein is involved in receptor-to-channel coupling.