the-xenopus-oocyte-cut-open-vaseline-gap-voltage-clamp-technique-with

The <em>Xenopus</em> Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

The cardiac cell is a complex biological system where various processes interact to generate electrical excitation (the action potential, AP) and contraction. During AP generation, membrane ion channels interact nonlinearly with dynamically changing ionic concentrations and varying transmembrane voltage, and are subject to regulatory processes. In recent years, a large body of knowledge has accumulated on the molecular structure of cardiac ion channels, their function, and their modification by genetic mutations that are associated with cardiac arrhythmias and sudden death. However, ion channels are typically studied in isolation (in expression systems or isolated membrane patches), away from the physiological environment of the cell where they interact to generate the AP. A major challenge remains the integration of ion-channel properties into the functioning, complex and highly interactive cell system, with the objective to relate molecular-level processes and their modification by disease to whole-cell function and clinical phenotype. In this article we describe how computational biology can be used to achieve such integration. We explain how mathematical (Markov) models of ion-channel kinetics are incorporated into integrated models of cardiac cells to compute the AP. We provide examples of mathematical (computer) simulations of physiological and pathological phenomena, including AP adaptation to changes in heart rate, genetic mutations in SCN5A and HERG genes that are associated with fatal cardiac arrhythmias, and effects of the CaMKII regulatory pathway and beta-adrenergic cascade on the cell electrophysiological function.

Computational biology in the study of cardiac ion channels and cell electrophysiology.

Most potassium channels are tetramers of four homologous polypeptides (subunits). During channel gating, each subunit undergoes several conformational changes independent of the state of other subunits before reaching a permissive state, from which the channel can open. However, transition from the permissive states to the open state involves a concerted movement of all subunits. This cooperative transition must be included in Markov models of channel gating. Previously, it was implemented by considering all possible combinations of four subunit states in a much larger expanded model of channel states (e.g., 27,405 channel states versus 64 subunit states), which complicates modeling and is computationally intense, especially when accurate modeling requires a large number of subunit states. To overcome these complexities and retain the tetrameric molecular structure, a modeling approach was developed to incorporate the cooperative transition directly from the subunit models. In this approach, the open state is separated from the subunit models and represented by the net flux between the open state and the permissive states. Dynamic variations of the probability of state residencies computed using this direct approach and the expanded model were identical. Implementation of the direct approach is simple and its computational time is orders-of-magnitude shorter than the equivalent expanded model.

Modeling subunit cooperativity in opening of tetrameric ion channels.

Computational models of cardiac myocytes are important tools for understanding ionic mechanisms of arrhythmia. This work presents a new model of the canine epicardial myocyte that reproduces a wide range of experimentally observed rate-dependent behaviors in cardiac cell and tissue, including action potential (AP) duration (APD) adaptation, restitution, and accommodation. Model behavior depends on updated formulations for the 4-aminopyridine-sensitive transient outward current (I(to1)), the slow component of the delayed rectifier K(+) current (I(Ks)), the L-type Ca(2+) channel current (I(Ca,L)), and the Na(+)-K(+) pump current (I(NaK)) fit to data from canine ventricular myocytes. We found that I(to1) plays a limited role in potentiating peak I(Ca,L) and sarcoplasmic reticulum Ca(2+) release for propagated APs but modulates the time course of APD restitution. I(Ks) plays an important role in APD shortening at short diastolic intervals, despite a limited role in AP repolarization at longer cycle lengths. In addition, we found that I(Ca,L) plays a critical role in APD accommodation and rate dependence of APD restitution. Ca(2+) entry via I(Ca,L) at fast rate drives increased Na(+)-Ca(2+) exchanger Ca(2+) extrusion and Na(+) entry, which in turn increases Na(+) extrusion via outward I(NaK). APD accommodation results from this increased outward I(NaK). Our simulation results provide valuable insight into the mechanistic basis of rate-dependent phenomena important for determining the heart's response to rapid and irregular pacing rates (e.g., arrhythmia). Accurate simulation of rate-dependent phenomena and increased understanding of their mechanistic basis will lead to more realistic multicellular simulations of arrhythmia and identification of molecular therapeutic targets.

Properties and ionic mechanisms of action potential adaptation, restitution, and accommodation in canine epicardium.

Ion-channel function is determined by its gating movement. Yet, molecular dynamics and electrophysiological simulations were never combined to link molecular structure to function. We performed multiscale molecular dynamics and continuum electrostatics calculations to simulate a cardiac K(+) channel (I(Ks)) gating and its alteration by mutations that cause arrhythmias and sudden death. An all-atom model of the I(Ks) alpha-subunit KCNQ1, based on the recent Kv1.2 structure, is used to calculate electrostatic energies during gating. Simulations are compared with experiments where varying degrees of positive charge-added via point mutation-progressively reduce current. Whole-cell simulations show that mutations cause action potential and ECG QT interval prolongation, consistent with clinical phenotypes. This framework allows integration of multiscale observations to study the molecular basis of excitation and its alteration by disease.

A multiscale model linking ion-channel molecular dynamics and electrostatics to the cardiac action potential.

Ionenkanal Funktion wird durch seine gating Bewegung bestimmt. Dennoch wurden Molekulare Dynamik und elektrophysiologische Simulationen nie kombiniert, um molekulare Struktur mit Funktion verkn&uuml;pfen. Wir haben Multiskalen Molekulardynamik und Kontinuum Elektrostatik Berechnungen simulieren einen kardialen K(+)-Kanal (I(Ks)) Anschnitt- und ihrer Ver&auml;nderung durch Mutationen, die zu Herzrhythmusst&ouml;rungen und pl&ouml;tzlichen Herztod f&uuml;hren. Ein All-Atom-Modell von der I(Ks) Alpha-Untereinheit KCNQ1, basierend auf der j&uuml;ngsten Kv1.2-Struktur wird verwendet, um die elektrostatische Energie w&auml;hrend Anspritzung berechnen. Simulationen werden mit Experimenten verglichen, wo unterschiedliche Grade der positiven Ladung-&uuml;ber hinzugef&uuml;gt Punktmutation schrittweise Strom reduzieren. Ganze Zelle Simulationen zeigen, dass Mutationen Aktionspotential und ECG QT-Intervall-Verl&auml;ngerung, mit klinischen Ph&auml;notypen verursachen. Dieser Rahmen erm&ouml;glicht die Integration der Multiskalen Beobachtungen, die molekulare Grundlage der Erregung und ihre Ver&auml;nderung durch Krankheit zu studieren.

Ein Multiskalen-Modell Ionenkanal Molekulare Dynamik und Elektrostatik mit der kardialen Aktionspotential verkn&uuml;pfen.

Multi-scale electrophysiology modeling: from atom to organ.

The inherited channelopathies are a rare, heterogeneous group of diseases with widely variable clinical presentations and courses. Systematic clinical and experimental work has led to identification of disease-causing genetic mutations and their biophysical manifestation. The process by which the knowledge base is developed, from genetic mutation, to cardiac myocyte, to whole heart, and finally to clinical presentation, has dramatically expanded our understanding of these diseases. Most importantly, we can now begin to comprehend how small changes at the genetic level can dramatically influence a patient's clinical course.

Updates on the inherited cardiac ion channelopathies: from cell to clinical.

Ion channel associated diseases: overview of molecular mechanisms.

The number of voltage-gated sodium (Na(V)) channels available to generate action potentials in muscles and nerves is adjusted over seconds to minutes by prior electrical activity, a process called slow inactivation (SI). The basis for SI is uncertain. Na(V) channels have four domains (DI-DIV), each with a voltage sensor that moves in response to depolarizing stimulation over milliseconds to activate the channels. Here, SI of the skeletal muscle channel Na(V)1.4 is induced by repetitive stimulation and is studied by recording of sodium currents, gating currents, and changes in the fluorescence of probes on each voltage sensor to assess their movements. The magnitude, voltage dependence, and time course of the onset and recovery of SI are observed to correlate with voltage-sensor movements 10,000-fold slower than those associated with activation. The behavior of each voltage sensor is unique. Development of SI over 1-160 s correlates best with slow immobilization of the sensors in DI and DII; DIII tracks the onset of SI with less fidelity. Showing linkage to the sodium conduction pathway, pore block by tetrodotoxin affects both SI and immobilization of all the sensors, with DI and DII significantly suppressed. Recovery from SI correlates best with slow restoration of mobility of the sensor in DIII. The findings suggest that voltage-sensor movements determine SI and thereby mediate Na(V) channel availability.

Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels I: wild-type skeletal muscle Na(V)1.4.

In skeletal muscle, slow inactivation (SI) of Na(V)1.4 voltage-gated sodium channels prevents spontaneous depolarization and fatigue. Inherited mutations in Na(V)1.4 that impair SI disrupt activity-induced regulation of channel availability and predispose patients to hyperkalemic periodic paralysis. In our companion paper in this issue (Silva and Goldstein. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210909), the four voltage sensors in Na(V)1.4 responsible for activation of channels over microseconds are shown to slowly immobilize over 1-160 s as SI develops and to regain mobility on recovery from SI. Individual sensor movements assessed via attached fluorescent probes are nonidentical in their voltage dependence, time course, and magnitude: DI and DII track SI onset, and DIII appears to reflect SI recovery. A causal link was inferred by tetrodotoxin (TTX) suppression of both SI onset and immobilization of DI and DII sensors. Here, the association of slow sensor immobilization and SI is verified by study of Na(V)1.4 channels with a hyperkalemic periodic paralysis mutation; L689I produces complex changes in SI, and these are found to manifest directly in altered sensor movements. L689I removes a component of SI with an intermediate time constant (~10 s); the mutation also impedes immobilization of the DI and DII sensors over the same time domain in support of direct mechanistic linkage. A model that recapitulates SI attributes responsibility for intermediate SI to DI and DII (10 s) and a slow component to DIII (100 s), which accounts for residual SI, not impeded by L689I or TTX.

Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: a periodic paralysis mutation in Na(V)1.4 (L689I).

Voltage-gated ion channels generate dynamic ionic currents that are vital to the physiological functions of many tissues. These proteins contain separate voltage-sensing domains, which detect changes in transmembrane voltage, and pore domains, which conduct ions. Coupling of voltage sensing and pore opening is critical to the channel function and has been modeled as a protein-protein interaction between the two domains. Here, we show that coupling in Kv7.1 channels requires the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). We found that voltage-sensing domain activation failed to open the pore in the absence of PIP2. This result is due to loss of coupling because PIP2 was also required for pore opening to affect voltage-sensing domain activation. We identified a critical site for PIP2-dependent coupling at the interface between the voltage-sensing domain and the pore domain. This site is actually a conserved lipid-binding site among different K(+) channels, suggesting that lipids play an important role in coupling in many ion channels.

Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening.

The concept that pore-forming Kir6.2 and regulatory SUR2A subunits form cardiac ATP-sensitive potassium (K(ATP)) channels is challenged by recent reports that SUR1 is predominant in mouse atrial K(ATP) channels.

Article	Year
Computational biology in the study of cardiac ion channels and cell electrophysiology. Quarterly reviews of biophysics\| PubMed ID: 16848931	2006
Modeling subunit cooperativity in opening of tetrameric ion channels. Biophysical journal\| PubMed ID: 18621838	2008
Properties and ionic mechanisms of action potential adaptation, restitution, and accommodation in canine epicardium. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 19168720	2009
A multiscale model linking ion-channel molecular dynamics and electrostatics to the cardiac action potential. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19549851	2009
Multi-scale electrophysiology modeling: from atom to organ. The Journal of general physiology\| PubMed ID: 20513759	2010
Updates on the inherited cardiac ion channelopathies: from cell to clinical. Current treatment options in cardiovascular medicine\| PubMed ID: 22865245	2012
Ion channel associated diseases: overview of molecular mechanisms. Chemical reviews\| PubMed ID: 23151230	2012
Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels I: wild-type skeletal muscle Na(V)1.4. The Journal of general physiology\| PubMed ID: 23401571	2013
Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: a periodic paralysis mutation in Na(V)1.4 (L689I). The Journal of general physiology\| PubMed ID: 23401572	2013
Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23861489	2013
Heterogeneity and function of K(ATP) channels in canine hearts. Heart rhythm : the official journal of the Heart Rhythm Society\| PubMed ID: 23871704	2013

Jonathan R. Silva

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