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L Lacinová

Publications and source records attributed to L Lacinová.

At least 19 recordsLinked to original sources
PubMed2006

Gating of the expressed T-type Cav3.1 calcium channels is modulated by Ca2+.

AIM: We have investigated the influence of Ca2+ ions on the basic biophysical properties of T-type calcium channels. METHODS: The Cav3.1 calcium channel was transiently expressed in HEK 293 cells. Current was measured using the whole cell patch clamp technique. Ca2+ or Na+ ions were used as charge carriers. The intracellular Ca2+ was either decreased by the addition of 10 mm ethyleneglycoltetraacetic acid (EGTA) or increased by the addition of 200 microm Ca2+ into the non-buffered intracellular solution. Various combinations of extra- and intracellular solutions yielded high, intermediate or low intracellular Ca2+ levels. RESULTS: The amplitude of the calcium current was independent of intracellular Ca2+ concentrations. High levels of intracellular Ca2+ accelerated significantly both the inactivation and the activation time constants of the current. The replacement of extracellular Ca2+ by Na+ as charge carrier did not affect the absolute value of the activation and inactivation time constants, but significantly enhanced the slope factor of the voltage dependence of the inactivation time constant. Slope factors of voltage dependencies of channel activation and inactivation were significantly enhanced. The recovery from inactivation was faster when Ca2+ was a charge carrier. The number of available channels saturated for membrane voltages more negative than -100 mV for the Ca2+ current, but did not reach steady state even at -150 mV for the Na+ current. CONCLUSIONS: Ca2+ ions facilitate transitions of Cav3.1 channel from open into closed and inactivated states as well as backwards transition from inactivated into closed state, possibly by interacting with its voltage sensor.

Barium↗
PubMed2005

Voltage-dependent calcium channels.

Voltage-activated calcium channels can be divided into two subgroups based on their activation threshold, low-voltage-activated (LVA) and high-voltage-activated (HVA). Auxiliary subunits of the HVA calcium channels contribute significantly to biophysical properties of the channels. We have cloned and characterized members of two families of auxiliary subunits: alpha2delta and gamma. Two new alpha2delta subunits, alpha2delta-2 and alpha2delta-3, regulate all classes of HVA calcium channels. While the ubiquitous alpha2delta-2 modulates both neuronal and non-neuronal channels with similar efficiency, the alpha2delta-3 subunit regulates Ca(v)2.3 channels more effectively. Furthermore, alpha2delta-2 may modulate the LVA Ca(v)3.1 channel. Four new gamma subunits, gamma-2, gamma-3, gamma-4 and gamma-5, were characterized. The gamma-2 subunit modulated both the non-neuronal Ca(v)1.2 channel and the neuronal Ca(v)2.1 channel. The gamma-4 subunit affected only the Ca(v)2.1 channel. The gamma-5 subunit may be a regulatory subunit of the LVA Ca(v)3.1 channel. The Ca(v)1.2 channel is a major target for treatment of cardiovascular diseases. We have mapped the interaction site for clinically important channel blockers - dihydropyridines (DHPs) - and analysed the underlying inhibition mechanism. High-affinity inhibition is characterized by interaction with inactivated state of the channel. Its structural determinants are amino acids of the IVS6 segment, with smaller contribution of the IS6 segment, which contributes to voltage-dependence of DHP inhibition. Removal of amino acids responsible for the high-affinity inhibition revealed a low-affinity open channel block, in which amino acids of the IIIS5 and IIIS6 segments take part. Experiments with a permanently charged DHP suggested that there is another low-affinity interaction site on the alpha(1) subunit. We have cloned and characterized murine neuronal LVA Ca(v)3.1 channel. The channel has high sensitivity to the organic blocker mibefradil, moderate sensitivity to phenytoin, and low sensitivity to ethosuximide, amiloride and valproat. The channel is insensitive to tetrodotoxin and DHPs. The inorganic blockers Ni2+ and Cd2+ are moderately effective compared to La3+. The current through the Ca(v)3.1 channel inactivates faster with Ba2+ compared to Ca2+. Molecular determinants of fast inactivation are located in amino side of the intracellular carboxy terminus. The voltage dependence of charge movement is very shallow compared to the voltage dependence of current activation. Transfer of 30 % of charge correlates with activation of 70 % of measurable macroscopic current. Prolonged depolarization does not immobilize charge movement of the Ca(v)3.1 channel.

Animals↗
PubMed2004

Pharmacology of recombinant low-voltage activated calcium channels.

Several types of voltage- or ligand-activated calcium channels contribute to the excitability of neuronal cells. Low-voltage-activated (LVA), T-type calcium channels are characterised by relatively negative threshold of activation and therefore they can generate low-threshold spikes, which are essential for burst firing. At least three different proteins form T-type calcium current in neurons: Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3. Expression of these proteins in various brain regions is complementary. Individual channel types could be distinguished by different sensitivity towards inorganic cations. This inhibition can contribute to the toxicity of some heavy metals. Selective inhibition of T-type calcium channels by organic blockers may have clinical importance in some forms of epilepsy. Mibefradil inhibits the expressed Ca(v2)3.1, Ca(v)3.2 and Ca(v)3.3 channels in nanomolar concentrations with Ca(v)3.3 channel having lowest affinity. The sensitivity of the expressed Ca(v)3.1 channel to the antiepileptic drugs, valproate and ethosuximide, is low. Ca(v)3.1 channel is moderately sensitive to phenytoin. The Ca(v)3.2 channel is sensitive to ethosuximide, amlodipine and amiloride. All three LVA calcium channels are moderately sensitive to active metabolites of methosuximide, i.e. alpha-methyl-alpha-phenylsuccinimide. Several neuroleptics inhibit all three LVA channels in clinically relevant concentrations. All three channels are also inhibited by the endogenous cannabinoid anandamide. A high affinity peptide blocker for these Ca channels is the scorpion toxin kurtoxin which inhibits the Ca(v)3.1 and Ca(v)3.2, but not the Ca(v)3.3 channel in nanomolar concentrations. Nitrous oxide selectively inhibits the Ca(v)3.2, but not the Ca(v)3.1 channel. The Ca(v)3.2, but not the Ca(v)3.1 channel is potentiated by stimulation of Ca(2+)/CaM-dependent protein kinase.

Action Potentials↗
PubMed2003

Inhibition of Cav3.1 channel by silver ions.

We have investigated the effects of AgCl and AgNO3 on the Cav3.1 calcium channels stably expressed in the HEK 293 cells. Ca2+ was used as a charge carrier. Both forms of Ag+ blocked the Cav3.1 channel and negatively shifted the I-V relations in a concentration-dependent manner. The inhibition of current amplitude by AgCl was voltage-dependent and increased with increasing amplitude of the depolarizing pulse. Furthermore, AgCl but not AgNO3 accelerated the kinetics of current activation. No effect on current inactivation or steady-state inactivation of the channel was observed for AgCl or AgNO3.

Calcium Channels, L-Type↗
PubMed2000

Regulation of the calcium channel alpha(1G) subunit by divalent cations and organic blockers.

The pharmacological properties of the expressed murine T-type alpha(1G) channel were characterized using the whole cell patch clamp configuration. Ba(2+) or Ca(2+) were used as charge carriers. Both I(Ba) and I(Ca) were blocked by Ni(2+) and Cd(2+) with IC(50) values of 0.47+/-0.04 and 1.13+/-0.06 mM (Ni(2+)) and 162+/-13 and 658+/-23 microM (Cd(2+)), respectively. Ni(2+), but not Cd(2+), modified the gating of channel activation. Ni(2+) consistently accelerated channel deactivation while Cd(2+) had a similar effect only on I(Ca). The alpha(1G) channel was potently blocked by mibefradil in a dose- and voltage-dependent manner. I(Ba) was moderately blocked by phenytoin (IC(50) 73.9+/-1.9 microM) and was resistant to the block by valproate. Also 3 mM ethosuximide blocked 20 and 35% of the I(Ba) at a HP of -100 and -60 mV, respectively, while 5 mM amiloride inhibited I(Ba) by 38% and significantly slowed current activation. The alpha(1G) channel was not affected by 10 microM tetrodotoxin. Both 1 microM (+)isradipine and 10 microM nifedipine inhibited 18 and 14% of I(Ba) amplitude at a HP of -100 mV, and 23% and 29% of I(Ba) amplitude at a HP of -60 mV, respectively. The alpha(1G) current was minimally activated by 1 microM Bay K 8644.

Cadmium↗
PubMed2000

A family of gamma-like calcium channel subunits.

The gamma subunit was initially identified as an auxiliary subunit of the skeletal muscle calcium channel complex. Evidence for the existence of further gamma subunits arose following the characterization of a genetic defect that induces epileptic seizures in stargazer mice. We present here the first account of a family of at least five putative gamma subunits that are predominantly expressed in brain. The gamma-2 and gamma-4 subunits shift the steady-state inactivation curve to more hyperpolarized potentials upon coexpression with the P/Q type alpha(1A) subunit. The coexpression of the gamma-5 subunit accelerates the time course of current activation and inactivation of the alpha(1G) T-type calcium channel.

Amino Acid Sequence↗
PubMed2000

State- and isoform-dependent interaction of isradipine with the alpha1C L-type calcium channel.

We investigated the dihydropyridine (DHP) inhibition of barium current (I(Ba)) through the smooth muscle alpha1Ch and cardiac alpha1Ca splice variants of the L-type calcium channel using a whole-cell patch-clamp method. IC50 values for inhibition of current amplitude of the alpha1Cb channel were three to fivefold lower than for the alpha1Ca channel at holding potentials between -80 mV and -30 mV. No difference was found in either the transition of the channels into an inactivated state in the absence or presence of drug, or in the recovery from inactivation under control conditions. However, isradipine slowed the recovery from inactivation of alpha1Ca more effectively than alpha1Cb. To evaluate the interaction of isradipine with the open channel state of both splice variants, interactions with the inactivated state were selectively suppressed by the mutation of three amino acids in the IVS6 segment (Y1485I, M1486F, I1493L) in alpha1CbCh30 and alpha1CaCh30 channels. The extent of this interaction was seen by an acceleration of current decay. This was found to be identical for both splice variants. Our results suggest that the higher DHP selectivity of the alpha1Cb versus the alpha1Ca channel is caused by the structural difference in the binding site and not by different transitions between resting, open and inactivated states.

Alternative Splicing↗
PubMed2000

Low voltage activated calcium channels: from genes to function.

Cloning of three members of low-voltage-activated (LVA) calcium channel family, predominantly neuronal alpha1G and alpha1I, and ubiquitous alpha1H, enabled to investigate directly their electrophysiological and pharmacological profile as well as their putative subunit composition. All the three channels are half-activated at membrane potential about -40 mV and half-inactivated at about -70 mV. Kinetics of alpha1G and alpha1H channels activation and inactivation are similar and faster than that of alpha1I channel. All the three channels are blocked with high affinity by the organic blocker mibefradil. Another high affinity blocker is kurtoxin. Cloned LVA channels are relatively insensitive to antiepileptics, dihydropyridines and omega-conotoxins. Ni2+ is high affinity blocker of alpha1H channel only. Amiloride inhibits the alpha1H channel. The subunit composition of LVA channel remains unclear. Out of known high-voltage-activated calcium channel subunits, alpha2delta-2 and gamma-5 subunits significantly and systematically modified activation and/or inactivation of the current. In contrast, alpha2delta-1, alpha2delta-3, gamma-2 and gamma-4 subunits failed to modulate the current or had only minor effects.

Alternative Splicing↗
PubMed1999

Reconstruction of the dihydropyridine site in a non-L-type calcium channel: the role of the IS6 segment.

Mutations of eight to nine amino acids of IIIS5, IIIS6 and IVS6 segments were shown to reconstruct the dihydropyridine (DHP) interaction site in the non-L-type alpha1E or alpha1A calcium channels. The reconstructed site enabled enantiomer-selective inhibition and activation of the expressed chimeras by DHPs but failed to transfer voltage dependence of the current inhibition. Here we show that transfer of four non-conserved amino acids from the IS6 segment to the DHP-sensitive alpha1E chimera increased the inhibition by (+)isradipine at the hyperpolarized membrane potential of -100 mV and enhanced the voltage-dependent block.

Animals↗
PubMed1999

Absence of modulation of the expressed calcium channel alpha1G subunit by alpha2delta subunits.

1. The modulatory action of the alpha2delta subunit on various high-voltage-activated calcium channels has been demonstrated previously. However, very little is known about auxiliary subunit modulation of low-voltage-activated (LVA) calcium channels. We have examined the modulation of the alpha1G subunit corresponding to the neuronal T-type calcium channel by the ubiquitously expressed alpha2delta-1 and brain-specific alpha2delta-3 subunits. 2. The alpha1G subunit was expressed alone or in combination with either the alpha2delta-1 or alpha2delta-3 subunit in human embryonic kidney (HEK 293) cells and whole-cell barium currents were measured. The current density-voltage relationships for peak and sustained current, kinetics of current activation and inactivation, voltage dependence of current inactivation and time course of the recovery from inactivation were analysed for each type of expressed channel. No significant difference was found for any of the examined parameters. 3. These results suggest that the LVA alpha1G channel is not regulated by known auxiliary alpha2delta subunits.

Animals↗
PubMed1999

Molecular diversity of the calcium channel alpha2delta subunit.

Sequence database searches with the alpha2delta subunit as probe led to the identification of two new genes encoding proteins with the essential properties of this calcium channel subunit. Primary structure comparisons revealed that the novel alpha2delta-2 and alpha2delta-3 subunits share 55.6 and 30.3% identity with the alpha2delta-1 subunit, respectively. The number of putative glycosylation sites and cysteine residues, hydropathicity profiles, and electrophysiological character of the alpha2delta-3 subunit indicates that these proteins are functional calcium channel subunits. Coexpression of alpha2delta-3 with alpha1C and cardiac beta2a or alpha1E and beta3 subunits shifted the voltage dependence of channel activation and inactivation in a hyperpolarizing direction and accelerated the kinetics of current inactivation. The kinetics of current activation were altered only when alpha2delta-1 or alpha2delta-3 was expressed with alpha1C. The effects of alpha2delta-3 on alpha1C but not alpha1E are indistinguishable from the effects of alpha2delta-1. Using Northern blot analysis, it was shown that alpha2delta-3 is expressed exclusively in brain, whereas alpha2delta-2 is found in several tissues. In situ hybridization of mouse brain sections showed mRNA expression of alpha2delta-1 and alpha2delta-3 in the hippocampus, cerebellum, and cortex, with alpha2delta-1 strongly detected in the olfactory bulb and alpha2delta-3 in the caudate putamen.

Amino Acid Sequence↗
PubMed1999

A T-type calcium channel from mouse brain.

A member of the low-voltage-activated calcium channel family was identified in mouse brain by taking advantage of amino acid sequences that have been evolutionary conserved. The identified sequence is similar to that of the recently cloned rat alpha1G T-type calcium channel, but there are differences in two insertions in the intracellular connecting loops. Northern blot analysis indicates that its expression is strong in the brain. In situ hybridization revealed that, in mouse brain, the alpha1G mRNA is found in the cerebellum, hippocampus, thalamus and olfactory bulb. In contrast to L-type calcium channel currents, IBa and ICa through the alpha1G channel expressed in HEK293 cells did not differ in terms of current density, voltage dependence of current activation, inactivation and deactivation, and speed of recovery from voltage-dependent inactivation. The kinetics of ICa inactivation were significantly slower than those of IBa. The expressed alpha1G channel has a relatively high sensitivity to mibefradil, but is only slightly affected by Ni2+.

Amino Acid Sequence↗
PubMed1999

Distinctions in the molecular determinants of charged and neutral dihydropyridine block of L-type calcium channels.

We investigated block of the alpha1Cb subunit of L-type calcium channels by dihydropyridines (DHPs) in which a permanently charged or neutral head group was linked to the active DHP moiety by a spacer chain containing ten methylene (-CH2) groups. We compared the sensitivity of channel modulation by the charged (DHPch) and neutral (DHPn) forms to specific alpha1Cb mutations in domains IIIS5, IIIS6, and IVS6, which had previously been shown to reduce channel modulation by the neutral DHP (+)-isradipine. The effects of these mutations were studied on channel block recorded from polarized (-80 mV) and depolarized (-40 mV) holding potentials (HPs). We found that channel block by DHPn was markedly reduced at both HPs by each mutation studied. In contrast, channel block by DHPch was only modestly reduced by mutations in IIIS6 and IVS6 for block from either -40 mV or -80 mV. Replacement of IIIS5 Thr1061 by Tyr, which abolished block by DHPn in an HP-independent manner, had little effect on channel block by DHPch recorded from -40 mV. However, this mutation markedly reduced DHPch block of currents recorded from a -80 mV HP. Inhibition of current by DHPch was not markedly use-dependent, in contrast with block by verapamil, another charged calcium channel blocker. These results suggest that the presence of a permanently charged head group restricts the access of the attached DHP moiety to a subset of interaction residues on the alpha1C subunit in a voltage-dependent manner. Furthermore, these restricted interactions confer distinct functional properties upon the charged DHP molecules.

Amino Acid Sequence↗
PubMed1998

Isradipine interacts with the open state of the L-type calcium channel at high concentrations.

Triple mutation of Tyr1485, Met1486 and Ile1493 in the IVS6 segment of alpha 1C-b subunit of the L-type calcium channel results in a loss of the high affinity inhibition by isradipine. The mutant channel (Ch30) yet exhibits a concentration-dependent inhibition by isradipine with a 110-fold lower affinity. The mechanisms underlying the remaining low affinity block were investigated. Isradipine accelerated the current decay in Ch30 but not in wild type channel in a concentration dependent manner. Dependence of the current amplitude inhibition on holding potential was parallel in Ch30 and in wild type channels, while the acceleration of current decay in Ch30 was independent of the membrane potential. The recovery from voltage-dependent inactivation was biphasic in both channels and was slowed down by isradipine in the wild type but not in the Ch30 channel. The change of the charge carrier (Ba2+ or Ca2+) and calcium chelator (EGTA or BAPTA) did not affect the acceleration of current decay indicating that isradipine did not interact with the Ca(2+)-inactivated state of the channel. These results demonstrate that the mutations of Ch30 affect selectively the high affinity inhibition of an inactivated channel and unmask a low affinity interaction of isradipine with an open state of the channel.

Barium↗
PubMed1996

The IVS6 segment of the L-type calcium channel is critical for the action of dihydropyridines and phenylalkylamines.

The current through the L-type calcium channel is inhibited and stimulated by distinct dihydropyridines at very low concentrations. The molecular determinants for the high affinity block and stimulation were investigated using chimeras between the class C and E calcium channels. Mutation of three amino acids in the last putative transmembrane segment (IVS6) of the alpha1C subunit decreased the affinity for (+)isradipine 100-fold without significantly affecting the basic properties of the expressed channel. Mutation of two of these three amino acids completely abolished the stimulatory effect of the calcium channel agonist Bay K 8644. These mutations only slightly affected the blocking efficacy of mibefradil and the phenylalkylamine devapamil. Three distinct but adjacently located amino acids mediated the high affinity block by devapamil. These results suggest that the IVS6 segment of the alpha1C subunit is critical for the high affinity interaction between the L-type calcium channel and the calcium channel agonist Bay K 8644 and the two antagonists isradipine and devapamil.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

The block of the expressed L-type calcium channel is modulated by the beta 3 subunit.

The alpha 1C subunit of the L-type calcium channel was stable, expressed alone or in combination with the beta 3 subunit in Chinese hamster ovary cells. The beta 3 subunit enhanced significantly the inactivation of barium currents indicating that both subunits interacted with each other. The beta 3 subunit decreased significantly the half-maximal inhibitory concentration of the calcium channel blockers (-)-gallopamil and verapamil, but did not affect significantly the block caused by isradipine and mibefradil at the holding potentials of -80 mV and -40 mV. These results suggest that the beta 3 subunit affects distinctly the interaction of the expressed alpha 1C subunit with different classes of organic calcium channel blockers.

Animals↗
PubMed1995

Interaction of Ro 40-5967 and verapamil with the stably expressed alpha 1-subunit of the cardiac L-type calcium channel.

The interaction of the nondihydropyridine calcium channel antagonist Ro 40-5967 with the stably expressed class C alpha 1-subunit of the cardiac L-type calcium channel was investigated and compared with that of verapamil by using the whole cell patch clamp configuration. Both compounds blocked the Ba++ inward current. The IC50 values at a holding potential of -80 or -40 mV were 4.9 and 1.4 microM for Ro 40-5967 and 250 and 15.5 microM for verapamil. Both Ro 40-5967 and verapamil induced a partial tonic block at a holding potential of -80 mV. The block increased with high depolarization rates. Both Ro 40-5967 and verapamil shifted the steady-state inactivation curve by more than 20 mV to hyperpolarized membrane potentials and decreased the inactivation rate constant. The effect of Ro 40-5967, but not that of verapamil, was attenuated by intracellular dialysis with GTP gamma S. The affinity for verapamil was not affected by replacing Ba++ by Ca++, but was increased by the coexpression of the beta 3-subunit. These results indicate that both compounds interact with high affinity with the inactivated channel state, but may interact additionally with the open channel.

Animals↗