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
• Názvy látek
• iontové kanály 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 voltage-dependent 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
• 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
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• iontové kanály MeSH
• vápníkové kanály - typ T 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 přístrojové vybavení   metody   MeSH
• design vybavení MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové potenciály fyziologie   MeSH
• metoda terčíkového zámku přístrojové vybavení   metody   MeSH
• neurony aferentní fyziologie   MeSH
• nízká teplota * MeSH
• perfuze přístrojové vybavení   MeSH
• prostředí kontrolované MeSH
• vysoká teplota * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

• MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• draslík metabolismus   MeSH
• draslíkové kanály metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• metoda terčíkového zámku MeSH
• proteiny přenášející kationty * MeSH
• Schizosaccharomyces pombe - proteiny * MeSH
• Schizosaccharomyces genetika   metabolismus   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• draslík MeSH
• draslíkové kanály MeSH
• membránové proteiny MeSH
• proteiny přenášející kationty * MeSH
• Schizosaccharomyces pombe - proteiny * MeSH
• transportní proteiny MeSH
• trk1 protein, S pombe MeSH Prohlížeč

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.

• Klíčová slova
• LFP, epilepsy, high frequency activity, in vitro, membrane chamber, patch clamp,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Ankyrin receptor subtype 1, Homology modeling, Molecular dynamics, Mutagenesis, S4–S5-linker, Transient receptor potential,
• 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
• kationtový kanál TRPA1 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
• Názvy látek
• 2,3,4-tri-O-acetylarabinopyranosyl isothiocyanate MeSH Prohlížeč
• asparagin MeSH
• isothiokyanatany MeSH
• kationtové kanály TRP MeSH
• kationtový kanál TRPA1 MeSH
• proteiny nervové tkáně MeSH
• serin MeSH
• TRPA1 protein, human MeSH Prohlížeč
• vápník MeSH
• vápníkové kanály MeSH

A versatile device for a patch-clamp amplifier is described. This device contains: (i) an acoustic indicator to monitor the input resistance of the patch pipette, which is used in search-mode to indicate the formation of seals; (ii) two pulse generators; and (iii) a staircase generator to produce various pulse and voltage step programs; (iv) a low-pass filter which is used to filter the output of the patch clamp amplifier; and (v) a remote control which is used to control the entire patch clamp experiment. This remote control is used to switch between search-, current clamp-, and voltage clamp-mode, to activate the respective stimulus potential programs, and to control the tape recorder. This electronic device can be easily connected to patch clamp amplifiers.

• MeSH
• elektrofyziologie přístrojové vybavení   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• buněčná membrána fyziologie   MeSH
• elektrofyziologie přístrojové vybavení   MeSH
• iontové kanály fyziologie   MeSH
• kuřecí embryo MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• iontové kanály MeSH

A variety of techniques of cell capacitance measurement have been proposed and applied in cellular electrophysiology. They are mostly based on the evaluation of membrane current responses to small changes in the membrane voltage. One of the currently used approaches applies the least-squares fit of an exponential current decay in response to voltage clamped rectangular pulses. In this study, we propose an alternative simpler approach to evaluation of the exponential parts in the current responses to square wave stimulation and present preliminary results of membrane capacitance evaluation. It is based on the property of the exponential function that has not yet been used to measure membrane capacitance. The time constant and the asymptote of the exponential waveform are unambiguously determined by the values read at three points separated by a constant time interval. In order to minimize the effect of noise and deviations from the exponential waveform, the triplet of points is designed to slide along the time axis. The results of the proposed approach and those previously evaluated by the least squares method are comparable. The method described may be advantageous for continuously recording changes in membrane capacitance.

• Klíčová slova
• Evaluation method, Membrane capacitance, Square wave stimulation,
• MeSH
• algoritmy MeSH
• elektrická kapacitance MeSH
• elektrická vodivost MeSH
• elektrofyziologie MeSH
• krysa rodu Rattus MeSH
• membránové potenciály fyziologie   MeSH
• metoda nejmenších čtverců MeSH
• metoda terčíkového zámku metody   MeSH
• modely neurologické MeSH
• neurony MeSH
• počítačová simulace MeSH
• reprodukovatelnost výsledků MeSH
• software MeSH
• srdeční síně patologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca2+ concentration ([Ca2+]i) remain unknown. We analyzed mechanisms regulating resting [Ca2+]i in dissociated rat melanotrophs by Ca2+-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca2+ channels (VGCCs) as well as removal of extracellular Ca2+ resulted in a rapid and reversible decrease in [Ca2+]i, indicating constitutive Ca2+ influx through L-type VGCCs. Reduction of extracellular Na+ concentration (replacement with NMDG+) similarly decreased resting [Ca2+]i. When cells were champed at -80 mV, decrease in the extracellular Na+ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na+ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K+ concentration was increased from 5 mM to 50 mM in the presence of K+ channel blockers, Ba2+ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca2+]i. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca2+]i in rat melanotrophs.

• Klíčová slova
• Ca(2+) homeostasis, Cation conductance, Patch-clamp, Ca(2+) imaging, ruthenium red, Rat melanotrophs, Voltage-gated calcium channels,
• MeSH
• kationtové kanály TRPV antagonisté a inhibitory   metabolismus   MeSH
• krysa rodu Rattus MeSH
• melanotropní buňky metabolismus   MeSH
• metoda terčíkového zámku MeSH
• potkani Wistar MeSH
• rutheniová červeň farmakologie   MeSH
• sodík metabolismus   MeSH
• vápník metabolismus   MeSH
• vápníkové kanály - typ L metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kationtové kanály TRPV MeSH
• rutheniová červeň MeSH
• sodík MeSH
• vápník MeSH
• vápníkové kanály - typ L MeSH