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.

Homer 1a Induces Calcium Channel Activation, but Does Not Change Their Properties in A431 Cells.

Store-operated channels activated in response to intracellular calcium store depletion represent the main pathway of calcium entry from the extracellular space in nonelectroexcitable cells. Adapter proteins organize the components of this system into integral complex. We studied the influence of adapter proteins of the Homer family on endogenous store-operated calcium Imin channels in A431 cells. Monomeric Homer 1a proteins increase activity of Imin channels, but did not modulate their electrophysiological properties. Recombinant Homer 1c protein did not block the induced calcium currents.

Electrophysiological Features of Single Store-Operated Calcium Channels in HEK S4 Cell Line with Stable STIM1 Protein Knockdown.

An important role in intracellular calcium signaling is played by store-operated channels activated by STIM proteins, calcium sensors of the endoplasmic reticulum. In stable STIM1 knockdown HEK S4 cells, single channels activated by depletion of intracellular calcium stores were detected by cell-attached patch-clamp technique and their electrophysiological parameters were described. Comparison of the properties of single channels in HEK293 and HEK S4 cells revealed no significant differences in their current-voltage curves, while regulation of store-operated calcium channels in these cell lines depended on the level of STIM1 expression. We can conclude that electrophysiological peculiarities of store-regulated calcium entry observed in different cells can be explained by differences in STIM1 expression.

[Histological characteristics of ocular structures after YAG-laser exposure of various sections of the crystalline lens in experimental studies]. / Gistologicheskaia kharakteristika struktur glaza posle IAG-lazernogo vozdeistviia na razlichnye otdely khrustalika v éksperimente.

This work was aimed at analysis of all the aspects, pathomorphologic included, of short-pulsed YAG laser exposure of the lens. Experiments were carried out with 79 rabbit eyes. After laser exposure the eyes were enucleated in 1-24 h and in 7-10 days. Thirty-one eyes were subjected to YAG laser ++capsule puncture. Fragmentation of intracapsular structures was carried out in 23 eyes. Combined operations were performed on 25 eyes. Every series comprised two groups, differing by the energy of laser exposure: 3-5 mJ. Histologic analysis has lead the authors to the following conclusions. The pattern and severity of pathomorphologic changes were directly related to the energy and time of laser exposure and site of the focal plane of irradiation. The major destructive shifts after YAG laser fragmentation of the cortical layer and capsulotomy are focussed at the site of exposure and involve the adjacent sections of the lens. Laser exposure of 3-5 mJ may be used to open the anterior capsule of the lens and to facilitate the cataract mass discharge in extracapsular cataract extraction. Use of combined method helps enhance induration and fragmentation of the cortical layers of the lens, rules out surgical discission of the anterior capsule of the lens, and facilitates removal of the nucleus and wash out of the lens mass.

[Effects of laser fragmentation of the lens mass on ocular tissues in experimental animals]. / Vliianie lazernoi fragmentatsii khrustalikovykh mass na tkani glaza v éksperimente.

Side effects of laser on the posterior corneal epithelium and the retina during intracapsular YAG laser fragmentation of transparent lens fibers in various modes of laser operation were experimentally studied. The pulse power varied from 3 to 7.7 mJ, number of pulses from 55 to 160, length of exposure 10(-8) sec, diameter of the spot in the focus 30 microns. Animal eyes were enucleated on days 3-6 after the procedure. Posterior corneal epithelium was examined in the Hitachi scanning electron microscope, the retina in a light microscope. The findings evidence that injury to the posterior corneal epithelium in intracapsular YAG laser fragmentation of the lens depends mostly on the number of pulses and not on the exposure energy. A safe number of coagulates should not surpass 100, this being quite sufficient for fragmentation of the lens fibers at exposure power of 5-6 mJ. Such operation mode is safe for the retina as well, this recommending it for clinical use.

[Method of lensectomy in high myopia]. / Metod lenséktomii pri blizorukosti vysokoi stepeni.

The authors suggest a method of removing a transparent lens in high myopia patients aged over 40 with a dense lens nucleus. The method consists of 3 stages. Stage I consists in preliminary intracapsular YAG laser fragmentation of lenticular fibrils, performed 2-4 days before surgery. Stage II consists in YAG laser anterior capsulotomy directly before lens extraction. Stage III consists in lenticular mass aspiration irrigation via 2 small incisions on the cornea. The described lensectomy method was used in 23 patients (44 eyes) aged 40 to 55 with refraction of 16.0 to 23.0 diopters. The time of lenticular mass aspiration was cut down by half as against that after lensectomy without preliminary intracapsular YAG laser fragmentation of lenticular fibrils. No complications during or after surgery were recorded. After surgery vision acuity improved by 13 times without correction and two times with correction.