Biomedical subjects

Katarzyna Mercik

Publications and source records attributed to Katarzyna Mercik.

10 recordsLinked to original sources

PubMed2006

Effect of extracellular pH on recombinant alpha1beta2gamma2 and alpha1beta2 GABAA receptors.

Recently, we have reported that extracellular protons allosterically modulated neuronal GABA(A) receptors [Mozrzymas, J.W., Zarnowska, E.D., Pytel, M., Mercik, K., 2003a. Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of desensitiztion process. Journal of Neuroscience 23, 7981-7992]. However, GABAARs in neurons are heterogeneous and the effect of hydrogen ions depends on the receptor subtype. In particular, gamma2 subunit sets the receptor sensibility to several modulators including protons. However, the mechanisms whereby protons modulate gamma2-containing and gamma2-free GABAARs have not been fully elucidated. To this end, current responses to ultrafast GABA applications were recorded for alpha1beta2gamma2 and alpha1beta2 receptors at different pH values. For both receptor types, increase in pH induced a decrease in amplitudes of currents elicited by saturating [GABA] but this effect was stronger for alpha1beta2 receptors. In the case of alpha1beta2gamma2 receptors, protons strongly affected the current time course due to a down regulation of binding and desensitization rates. This effect was qualitatively similar to that described in neurons. Protons strongly influenced the amplitude of alpha1beta2 receptor-mediated currents but the effect on their kinetics was weak suggesting a predominant direct non-competitive inhibition with a minor allosteric modulation. In conclusion, we provide evidence that extracellular protons strongly affect GABAA receptors and that, depending on the presence of the gamma2 subunit, the modulatory mechanisms show profound quantitative and qualitative differences.

Allosteric Regulation↗

PubMed2006

Interaction between cyclodextrin and neuronal membrane results in modulation of GABA(A) receptor conformational transitions.

Cyclodextrins (CDs) are nanostructures widely applied in biotechnology and chemistry. Owing to partially hydrophobic character, CDs interact with biological membranes. While the mechanisms of CDs interactions with lipids were widely studied, their effects on proteins are less understood. In the present study we investigated the effects of beta cyclodextrin (betaCD) on GABA(A) receptor (GABA(A)R) gating. To reliably resolve the kinetics of conformational transitions, currents were elicited by ultrafast gamma-aminobutyric acid (GABA) applications to outside-out patches from rat cultured hippocampal neurons. betaCD increased the amplitude of responses to saturating GABA concentration ([GABA]) in a dose-dependent manner and this effect was accompanied by profound alterations in the current kinetics. Current deactivation was slowed down by betaCD but this effect was biphasic with a maximum at around 0.5 mM betaCD. While the fast deactivation time constant was monotonically slowed down within considered betaCD concentration range, the slow component first increased and then, at millimolar betaCD concentration, decreased. The rate and extent of desensitization was decreased by betaCD in a dose-dependent manner. The analysis of current responses to nonsaturating [GABA] indicated that betaCD affected the GABA(A)R agonist binding site by slowing down the unbinding rate. Modulation of GABA(A)R desensitization and binding showed different concentration-dependence suggesting different modualtory sites with higher affinity of the latter one. All the betaCD effects were fully reversible indicating that cholesterol uptake into betaCD was not the primary mechanism. We conclude that betaCD is a strong modulator of GABA(A)R conformational transitions.

Animals↗

PubMed2005

The voltage dependence of GABAA receptor gating depends on extracellular pH.

Recent studies have indicated that changes in extracellular pH and in membrane voltage affect the gamma-amino-n-butyric acid type A receptor gating mainly by altering desensitization and binding. To test whether the effects of membrane potential and pH are additive, their combined actions were investigated. By analyzing the current responses to rapid gamma-amino-n-butyric acid applications, we found that the current to voltage relationship was close to linear at acid pH but the increasing pH induced an inward rectification. Desensitization was enhanced at depolarizing potentials, but this strongly depended on pH, being weak at acidic and strong at basic pH values. A similar trend was observed for the onset rate of responses to saturating gamma-amino-n-butyric acid concentration. These data provide evidence that the voltage sensitivity of GABAA receptors depends on extracellular pH.

Animals↗

PubMed2005

Membrane voltage modulates the GABA(A) receptor gating in cultured rat hippocampal neurons.

The kinetics of GABAergic currents in neurons is known to be modulated by the membrane voltage but the underlying mechanisms have not been fully explored. In particular, the impact of membrane potential on the GABA(A) receptor gating has not been elucidated. In the present study, the effect of membrane voltage on current responses elicited by ultrafast GABA applications was studied in cultured hippocampal neurons. The current to voltage relationship (I-V) for responses to saturating [GABA] (10 mM) showed an inward rectification (slope conductance at positive voltages was 0.62 +/- 0.05 of that at negative potentials). On the contrary, I-V for currents evoked by low [GABA] (1 microM) showed an outward rectification. The onset of currents elicited by saturating [GABA] was significantly accelerated at positive potentials. Analysis of currents evoked by prolonged applications of saturating [GABA] revealed that positive voltages significantly increased the rate and extent of desensitization. The onsets of current responses to non-saturating [GABA] were significantly accelerated at positive voltages indicating an enhancement of the binding rate. However, at low [GABA] at which the onset rate is expected to approach an asymptote set by opening/closing and unbinding rates, no significant modification of current onset by voltage was observed. Quantitative analysis based on model simulations indicated that the major effect of membrane depolarization was to increase the rates of binding, desensitization and of opening as well as to slightly reduce the rate of exit from desensitization. In conclusion, we provide evidence that membrane voltage affects the GABA(A) receptor microscopic gating.

Algorithms↗

PubMed2003

Recombinant alpha 1 beta 2 gamma 2 GABA(A) receptors expressed in HEK293 and in QT6 cells show different kinetics.

Neuronal gamma-aminobutyric acid (GABA)(A) receptors are extremely heterogeneous and therefore GABAergic currents represent responses of unknown mixture of receptor subtypes. Expression of recombinant receptors in foreign cells allows to investigate a defined receptor subtype but its properties can be altered due to, e.g., differences in the endogenous modulators. In the present study the alpha1beta2gamma2 receptors were expressed in HEK293 and QT-6 cells and current responses to ultrafast GABA applications were recorded. Rise time and rapid deactivation component were faster in responses recorded from QT-6 cells. Moreover, in QT-6 cells desensitization was faster and more profound. Recovery in the paired pulse experiments was faster in HEK293 cells. In conclusion, we provide evidence that recombinant receptors may show functional differences when expressed in different cells.

Cell Line↗

PubMed2003

Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of the desensitization process.

Protons are the most ubiquitous and very potent modulators of the biological systems. Hydrogen ions are known to modulate GABA(A) receptors (GABA(A)Rs), but the mechanism whereby these ions affect IPSCs and the gating of GABA(A)Rs is not clear. In the present study we examined the effect of protons on miniature IPSCs (mIPSCs) and found that hydrogen ions strongly affected both their amplitude and time course. To explore the underlying mechanisms with resolution adequate to the time scale of synaptic transmission, we recorded current responses to ultrafast GABA applications at various pH. These experiments revealed that the major effect of protons on GABA(A)R gating is a strong enhancement of desensitization and binding rates at increasing pH. This analysis also indicated that desensitization rate is the fastest ligand-independent transition in the GABA(A)R gating scheme. Although proton effects on the time course of mIPSCs and current responses to saturating [GABA] were similar, the pH dependencies of amplitudes were almost opposite. Our quantitative analysis, based on model simulations, indicated that this difference resulted from a much shorter receptor exposure to agonist in the case of mIPSCs. Modeling of IPSCs as current responses to brief exponentially decaying GABA applications was sufficient to reproduce correctly the pH dependence of mIPSCs, and optimal fit was obtained for peak [GABA] of 1.5-3 mm and a clearance time constant of 0.075-0.125 msec. Our analysis indicates that, for these parameters of GABA transient, in control conditions (pH 7.2) mIPSCs are not saturated.

Animals↗

PubMed2003

Binding sites, singly bound states, and conformation coupling shape GABA-evoked currents.

The time course of GABA-evoked currents is the main source of information on the GABA(A) receptor gating. Since the kinetics of these currents depends on the transitions between several receptor conformations, it is a major challenge to define the relations between current kinetics and the respective rate constants of the microscopic gating scheme. The aim of this study was to further explore the impact of different GABA(A) receptor conformations on the kinetics of currents elicited by ultra-fast GABA applications. We show that the rising phase and amplitude of GABA-evoked currents depend on desensitization and singly bound states. The occupancy of bound receptors depends not only on binding properties but also on opening/closing and desensitization. The impact of such functional coupling between channel states is critical in conditions of high non-equilibrium typical for synaptic transmission. The concentration dependence of the rising phase of the GABA-elicited current indicates positive cooperativity between agonist binding sites. We provide evidence that preequilibration at low GABA concentrations reduce GABA-evoked currents due to receptor trapping in a singly bound desensitized state.

Animals↗

PubMed2003

Resolving the ionotropic receptor kinetics and modulation in the time scale of synaptic transmission.

Synaptic transmission plays a crucial role in signal transduction in the adult central nervous system. It is known that synaptic transmission can be modulated by physiological and pathological processes and a number of factors including metal ions, pH, drugs, etc. The patch-clamp technique allows to measure postsynaptic currents, but the mechanism of these currents modulation remains unclear. The estimated value of neurotransmitter transient indicates that this time course is very short and the activation of postsynaptic receptors is extremely non-equilibrient. The ultrafast perfusion system makes it possible to mimic synaptic conditions and, additionally, the agonist concentration can be controlled, which is very important for pharmacokinetic studies. In the present paper, examples of pharmacological modulation of mIPSC kinetics and currents evoked by ultrafast agonist application are presented.

Cell Line↗

PubMed2002

Saturation and self-inhibition of rat hippocampal GABA(A) receptors at high GABA concentrations.

Current responses to ultrafast gamma-aminobutyric acid (GABA) applications were recorded from excised patches in rat hippocampal neurons to study the gating properties of GABA(A) receptors at GABA concentrations close to saturating ones and higher. The amplitude of currents saturated at approximately 1 mm, while the onset rate of responses reached saturation at 4-6 mm GABA. At high GABA concentrations (> 10 mm), the amplitude of current responses was reduced in a dose-dependent manner with a half-blocking GABA concentration of approximately 50 mm. The peak reduction at high GABA doses was accompanied by a tendency to increase the steady-state to peak ratio. At concentrations higher than 30 mm, this effect took the form of a rebound current, i.e. during the prolonged GABA applications, the current firstly declined due to desensitization onset and then, instead of decreasing towards a steady-state value, clearly increased. Both the self-inhibition of GABA(A) receptors by high GABA doses and rebound were clearly voltage dependent, being larger at positive holding potentials. The fast desensitization component accelerated with depolarization at all saturating [GABA] tested. The rebound phenomenon indicates that the self-block of GABAA receptors is state dependent, and suggests that the sojourn in the desensitized conformation provides a 'rescue' from the block. We propose that high GABA concentrations inhibit the receptors by direct occlusion of the channel pore having no effect on the receptor gating.

Animals↗

PubMed2002

[Effect of changes in extracellular pH on GABAA receptors in neurons].

Local pH values of both intra- and extracellular liquids can be regulated by a number of mechanisms including membrane transport and metabolism. It is known that the changes of extracellular pH accompanying physiological and pathological processes are sufficient to affect several important structures such as ionic channels, transporters, receptors etc. In particular, several reports indicate that GABAA receptor is strongly modulated by this factor. The effect of pH on these receptors strongly depend on the subtype of GABAA receptor (subunit composition). The application of ultrafast perfusion system allowed to explore the mechanisms of pH effect on GABAA receptors in neurons. It is concluded that changes in pH exert their effects by allosteric modulation of GABAA receptors.

Animals↗