patch-clamp technique.
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The patch clamp technique, developed in late 1970s, started a new period of experimental cardiac electrophysiology enabling measurement of ionic currents on isolated cardiomyocytes down to the level of single channels. Since that time, the technique has been substantially improved by development of several upgraded modifications providing so far unavailable data (e.g. action potential clamp, dynamic clamp, high-resolution scanning patch clamp), or facilitating the patch clamp technique by increasing its efficiency (planar patch clamp, automated patch clamp). The current review summarizes the leading new patch clamp based techniques used in cardiac cellular electrophysiology, their principles and prominent related papers.
- MeSH
- akční potenciály fyziologie MeSH
- design vybavení trendy MeSH
- gating iontového kanálu fyziologie MeSH
- iontové kanály metabolismus MeSH
- lidé MeSH
- membránové potenciály fyziologie MeSH
- metoda terčíkového zámku přístrojové vybavení trendy MeSH
- mikroelektrody trendy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Patch clamp method developed more than 30 years ago is widely used for investigation of cellular excitability manifested as transmembrane ionic current and/or generation of action potentials. This technique could be applied to measurement of ionic currents flowing through individual (single) ion channels or through the whole assembly of ion channels expressed in the whole cell. Whole cell configuration is more common for measurement of ion currents and the only one enabling measurement of action potentials. This method allows detailed analysis of mechanisms and structural determinants of voltagedependent gating of ion channels as well as regulation of channel activity by intracellular signaling pathways and pharmacological agents.
- MeSH
- akční potenciály MeSH
- biologické modely MeSH
- buněčná membrána metabolismus MeSH
- financování organizované MeSH
- gating iontového kanálu MeSH
- iontové kanály metabolismus MeSH
- lidé MeSH
- membránové potenciály MeSH
- metoda terčíkového zámku MeSH
- vápníková signalizace MeSH
- vápníkové kanály - typ T metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
We have developed an improved technique for fast cooling and heating of solutions superfusing isolated cells under patch-clamp or calcium imaging conditions. The system meets the requirements for studying temperature dependency of all kinds of ion channels, in particular temperature-gated ion channels. It allows the application of temperature changes within a range of 5-60 degrees C at maximum rates of -40 degrees C/s to 60 degrees C/s. Barrels filled with different solutions are connected to a manifold consisting of seven silica capillaries (320 microm inner diameter, i.d.). A common outlet consists of a glass capillary through which the solutions are applied onto the cell surface. The upper part of this capillary is embedded in a temperature exchanger driven by a miniature Peltier device which preconditions the temperature of the passing solution. The lower part of the capillary carries an insulated copper wire, densely coiled over a length of 7 mm, and connected to a dc current source for resistive heating. The Peltier device and the heating element are electrically connected to the headstage probe which is fixed on to a micromanipulator for positioning of the manifold. The temperature of the flowing solution is measured by a miniature thermocouple inserted into the common outlet capillary near to its orifice which is placed at a distance of less than 100 microm from the surface of the examined cell. The temperature is either manually controlled by voltage commands or adjusted via the digital-to-analog converter of a conventional data acquisition interface. Examples are given of using the device in patch-clamp studies on heterologously expressed TRPV1, TRPM8, and on cultured rat sensory neurons.
- MeSH
- akční potenciály fyziologie MeSH
- analýza selhání vybavení MeSH
- buněčné kultury metody přístrojové vybavení MeSH
- design vybavení MeSH
- financování organizované MeSH
- kultivované buňky MeSH
- lidé MeSH
- membránové potenciály fyziologie MeSH
- metoda terčíkového zámku metody přístrojové vybavení MeSH
- neurony aferentní fyziologie MeSH
- nízká teplota MeSH
- perfuze přístrojové vybavení MeSH
- prostředí kontrolované MeSH
- vysoká teplota MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- hodnotící studie MeSH
Highlights Simultaneous epileptiform LFPs and single-cell activity can be recorded in the membrane chamber.Interneuron firing can be linked to epileptiform high frequency activity.Fast ripples, unique to chronic epilepsy, can be modeled in ex vivo tissue from TeNT-treated rats. Traditionally, visually-guided patch clamp in brain slices using submerged recording conditions has been required to characterize the activity of individual neurons. However, due to limited oxygen availability, submerged conditions truncate fast network oscillations including epileptiform activity. Thus, it is technically challenging to study the contribution of individual identified neurons to fast network activity. The membrane chamber is a submerged-style recording chamber, modified to enhance oxygen supply to the slice, which we use to demonstrate the ability to record single-cell activity during in vitro epilepsy. We elicited epileptiform activity using 9 mM potassium and simultaneously recorded from fluorescently labeled interneurons. Epileptiform discharges were more reliable than in standard submerged conditions. During these synchronous discharges interneuron firing frequency increased and action potential amplitude progressively decreased. The firing of 15 interneurons was significantly correlated with epileptiform high frequency activity (HFA; ~100-500 Hz) cycles. We also recorded epileptiform activity in tissue prepared from chronically epileptic rats, treated with intrahippocampal tetanus neurotoxin. Four of these slices generated fast ripple activity, unique to chronic epilepsy. We showed the membrane chamber is a promising new in vitro environment facilitating patch clamp recordings in acute epilepsy models. Further, we showed that chronic epilepsy can be better modeled using ex vivo brain slices. These findings demonstrate that the membrane chamber facilitates previously challenging investigations into the neuronal correlates of epileptiform activity in vitro.
- MeSH
- elektrokortikografie MeSH
- epilepsie * diagnostické zobrazování MeSH
- interneurony MeSH
- krysa rodu rattus MeSH
- metoda terčíkového zámku * metody MeSH
- modely nemocí na zvířatech MeSH
- mozková kůra diagnostické zobrazování MeSH
- techniky in vitro metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- cystická fibróza patofyziologie MeSH
- lidé MeSH
- sodík patofyziologie MeSH
- techniky in vitro MeSH
- Check Tag
- lidé MeSH
The ankyrin transient receptor potential channel TRPA1 is a polymodal sensor for noxious stimuli, and hence a promising target for treating chronic pain. This tetrameric six-transmembrane segment (S1-S6) channel can be activated by various pungent chemicals, such as allyl isothiocyanate or cinnamaldehyde, but also by intracellular Ca(2+) or depolarizing voltages. Within the S4-S5 linker of human TRPA1, a gain-of-function mutation, N855S, was recently found to underlie familial episodic pain syndrome, manifested by bouts of severe upper body pain, triggered by physical stress, fasting, or cold. To clarify the structural basis for this channelopathy, we derive a structural model of TRPA1 by combining homology modeling, molecular dynamics simulations, point mutagenesis and electrophysiology. In the vicinity of N855, the model reveals inter-subunit salt bridges between E854 and K868. Using the heterologous expression of recombinant wild-type and mutant TRPA1 channels in HEK293T cells, we indeed found that the charge-reversal mutants E854R and K868E exhibited dramatically reduced responses to chemical and voltage stimuli, whereas the charge-swapping mutation E854R/K868E substantially rescued their functionalities. Moreover, mutation analysis of highly conserved charged residues within the S4-S5 region revealed a gain-of-function phenotype for R852E with an increased basal channel activity, a loss of Ca(2+)-induced potentiation and an accelerated Ca(2+)-dependent inactivation. Based on the model and on a comparison with the recently revealed atomic-level structure of the related channel TRPV1, we propose that inter-subunit salt bridges between adjacent S4-S5 regions are crucial for stabilizing the conformations associated with chemically and voltage-induced gating of the TRPA1 ion channel.
- MeSH
- asparagin genetika MeSH
- elektrická stimulace MeSH
- gating iontového kanálu účinky léků fyziologie MeSH
- HEK293 buňky MeSH
- isothiokyanatany farmakologie MeSH
- kationtové kanály TRP chemie genetika metabolismus MeSH
- lidé MeSH
- membránové potenciály genetika MeSH
- metoda terčíkového zámku MeSH
- molekulární modely * MeSH
- mutace genetika MeSH
- mutageneze MeSH
- proteiny nervové tkáně chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- serin genetika MeSH
- terciární struktura proteinů MeSH
- transfekce MeSH
- vápník metabolismus MeSH
- vápníkové kanály chemie genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
