methods-for-isolation-culture-functional-characterization-sinoatrial

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

University of Colorado Anschutz Medical Campus

In skeletal muscle, excitation-contraction (EC) coupling and retrograde signaling are thought to result from direct interactions between the ryanodine receptor (RyR1) and the alpha(1) subunit of the dihydropyridine receptor (alpha(1S)). Previous work has shown that the s53 region of alpha(1S) (residues 720-765 in the II-III loop) and regions R10 (1635-2636) and R9 (2659-3720) of RyR1 are involved in this signaling. Using the yeast two-hybrid system, we here report an interaction between s53 and the sR16 region of RyR1 (1837-2168, within R10), whereas no interaction was seen using upstream residues of the alpha(1S) II-III loop (s31, 666-709). The specificity of the s53-sR16 interaction was tested by using fragments of the cardiac RyR (RyR2) and DHPR (alpha(1C)) that correspond to sR16 and s53, respectively. No interaction was observed for sR16 x c53 (alpha(1C) 850-897), but weak interaction was occasionally observed for s53 x cR16 (RyR2 1817-2142). To test the functional significance of the s53 x sR16 interaction, we expressed in dyspedic myotubes a chimeric RyR (chimeraR16) in which sR16 was substituted for the corresponding region of RyR2. ChimeraR16 was found to mediate weak skeletal-type EC coupling. To test the necessity of sR16 sequence for coupling, we used "chimeraR16-rev," in which sR16 and a small upstream region of RyR1 were replaced by RyR2 sequence. ChimeraR16-rev did not differ from RyR1 in its ability to mediate EC coupling. Thus, interaction between residues 720-765 of alpha(1S) and residues 1837-2168 of RyR1 appears to contribute to but is not essential for EC coupling in skeletal muscle.

Identification of a region of RyR1 that participates in allosteric coupling with the alpha(1S) (Ca(V)1.1) II-III loop.

Spontaneous rhythmic activity in mammalian heart and brain depends on pacemaker currents (I(h)), which are produced by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here, we report that the mouse HCN2 pacemaker channel isoform also produced a large instantaneous current (I(inst(HCN2))) in addition to the well characterized, slowly activating I(h). I(inst(HCN2)) was specific to expression of HCN2 on the plasma membrane and its amplitude was correlated with that of I(h). The two currents had similar reversal potentials, and both were modulated by changes in intracellular Cl(-) and cAMP. A mutation in the S4 domain of HCN2 (S306Q) decreased I(h) but did not alter I(inst(HCN2)), and instantaneous currents in cells expressing either wild type HCN2 or mutant S306Q channels were insensitive to block by Cs(+). Co-expression of HCN2 with the accessory subunit, MiRP1, decreased I(h) and increased I(inst(HCN2)), suggesting a mechanism for modulation of both currents in vivo. These data suggest that expression of HCN channels may be accompanied by a background conductance in native tissues and are consistent with at least two open states of HCN channels: I(inst(HCN2)) is produced by a Cs(+)-open state; hyperpolarization produces an additional Cs(+)-sensitive open state, which results in I(h).

Pacemaker channels produce an instantaneous current.

In mammalian heart and brain, pacemaker currents are produced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which probably exist as heteromeric assemblies of different subunit isoforms. To investigate the molecular domains that participate in assembly and membrane trafficking of HCN channels, we have used the yeast two-hybrid system, patch clamp electrophysiology, and confocal microscopy. We show here that the N termini of the HCN1 and HCN2 isoforms interacted and were essential for expression of functional homo- or heteromeric channels on the plasma membrane of Chinese hamster ovary cells. We also show that the cyclic nucleotide binding domain (CNBD) of HCN2 was required for the expression of functional homomeric channels. This expression was dependent on a 12-amino acid domain corresponding to the B-helix in the CNBD of the catabolite activator protein. However, co-expression with HCN1 of an HCN2 deletion mutant lacking the CNBD rescued surface immunofluorescence and currents, indicating that a CNBD need not be present in each subunit of a heteromeric HCN channel. Furthermore, neither CNBDs nor other COOH-terminal domains of HCN1 and HCN2 interacted in yeast two-hybrid assays. Thus, interaction between NH(2)-terminal domains is important for HCN subunit assembly, whereas the CNBD is important for functional expression, but its absence from some subunits will still allow for the assembly of functional channels.

Different roles for the cyclic nucleotide binding domain and amino terminus in assembly and expression of hyperpolarization-activated, cyclic nucleotide-gated channels.

Despite permeability to both K(+) and Na(+), hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels contain the K(+) channel signature sequence, GYG, within the selectivity filter of the pore. Here, we show that this region is involved in regulating gating in a mouse isoform of the pacemaker channel (mHCN2). A mutation in the GYG sequence of the selectivity filter (G404S) had different effects on the two components of the wild-type current; it eliminated the slowly activating current (I(f)) but, surprisingly, did not affect the instantaneous current (I(inst)). Confocal imaging and immunocytochemistry showed G404S protein on the periphery of the cells, consistent with the presence of channels on the plasma membrane. Experiments with the wild-type channel showed that the rate of I(f) deactivation and I(f) amplitude had a parallel dependence on the ratio of K(+)/Na(+) driving forces. In addition, the amplitude of fully activated I(f), unlike I(inst), was not well predicted by equal and independent flow of K(+) and Na(+). The data are consistent with two separable gating mechanisms associated with pacemaker channels: one (I(f)) that is sensitive to voltage, to a mutation in the selectivity filter, and to driving forces for permeating cations and another (I(inst)) that is insensitive to these influences.

Separable gating mechanisms in a Mammalian pacemaker channel.

Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits. Previously, we suggested that the NH(2) termini of the mouse HCN2 isoform were important for subunit co-assembly and functional channel expression. Using an alignment strategy together with yeast two-hybrid assays, patch clamp electrophysiology, and confocal imaging, we have now identified a domain within the NH(2) terminus of the HCN2 subunit that is responsible for interactions between NH(2) termini and promoting the trafficking of functional channels to the plasma membrane. This domain is composed of 52 amino acids, is located adjacent to the putative first transmembrane segment, and is highly conserved among the mammalian HCN isoforms. This conserved domain, but not the remaining unconserved NH(2)-terminal regions of HCN2, specifically interacted with itself in yeast two-hybrid assays. Moreover, the conserved domain was important for expression of currents. Whereas relatively normal whole cell HCN2 currents were produced by channels containing only the conserved domain, further deletion of this region, leaving only a more polar and putative coiled-coil segment, eliminated HCN2 currents and resulted in proteins that localized predominantly in perinuclear compartments. Thus, we suggest that this conserved domain is the critical NH(2)-terminal determinant of subunit co-assembly and trafficking of pacemaker channels.

A conserved domain in the NH2 terminus important for assembly and functional expression of pacemaker channels.

Hyperpolarization-activated HCN channels are modulated by direct binding of cyclic nucleotides. For HCN2 channels, cAMP shifts the voltage dependence for activation, with relatively little change in the maximal conductance. By contrast, in spHCN channels, cAMP relieves a rapid inactivation process and produces a large increase in maximum conductance. Our results suggest that these two effects of cAMP represent the same underlying process. We also find that spHCN inactivation occurs not by closure of a specialized inactivation gate, as for other voltage-dependent channels, but by reclosure of the same intracellular gate opened upon activation. Effectively, the activation gate exhibits a "desensitization to voltage," perhaps by slippage of the coupling between the voltage sensors and the gate. Differences in the initial coupling efficiency could allow cAMP to produce either the inactivation or the shift phenotype by strengthening effective coupling: a shift would naturally occur if coupling is already strong in the absence of cAMP.

Inactivation in HCN channels results from reclosure of the activation gate: desensitization to voltage.

Hyperpolarization-activated HCN pacemaker channels are critical for the generation of spontaneous activity and the regulation of excitability in the heart and in many types of neurons. These channels produce both a voltage-dependent current (I(h)) and a voltage-independent current (I(inst) or VIC). In this study, we explored the molecular basis of the voltage-independent current. We found that for the spHCN isoform, VIC averaged approximately 4% of the maximum HCN conductance that could be activated by hyperpolarization. Cyclic AMP increased the voltage-independent current in spHCN to approximately 8% of maximum. In HCN2, VIC was approximately 2% of the maximal current, and was little affected by cAMP. VIC in both spHCN and HCN2 was blocked rapidly both by ZD7288 (an HCN channel blocker that is thought to bind in the conduction pore) and by application of Cd2+ to channels containing an introduced cysteine in the pore (spHCN-464C or HCN2-436C). These results suggest that VIC flows through the main conduction pathway, down the central axis of the protein. We suspected that VIC simply represented a nonzero limiting open probability for HCN channels at positive voltages. Surprisingly, we found instead that the spHCN channels carrying VIC were not in rapid equilibrium with the channels carrying the voltage-dependent current, because they could be blocked independently; a single application of blocker at a depolarized potential essentially eliminated VIC with little change in I(h). Thus, VIC appears to be produced by a distinct population of HCN channels. This voltage-independent current could contribute significantly to the role of HCN channels in neurons and myocytes; VIC flowing through the channels at physiological potentials would tend to promote excitability by accelerating both depolarization and repolarization.

Distinct populations of HCN pacemaker channels produce voltage-dependent and voltage-independent currents.

&#x4EA4;&#x611F;&#x795E;&#x7ECF;&#x7CFB;&#x7EDF;&#x901A;&#x8FC7;&#x6FC0;&#x6D3B; &beta; &#x80BE;&#x4E0A;&#x817A;&#x7D20;&#x80FD;&#x53D7;&#x4F53;&#x548C;&#x589E;&#x52A0;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x7AA6;&#x623F;&#x7ED3;&#x8425;&#x5730;&#x6C34;&#x5E73;&#x589E;&#x52A0;&#x5FC3;&#x7387;&#x3002;&#x8FD9;&#x79CD;&#x53CD;&#x5E94;&#x7684;&#x5206;&#x5B50;&#x57FA;&#x7840;&#x4E0D;&#x662F;&#x5F88;&#x4E86;&#x89E3; &#xFF1B;&#x7136;&#x800C;&#xFF0C;&#x5FC3;&#x810F;&#x6ED1;&#x7A3D;&#x5F53;&#x524D; &#xFF08;I(f)) &#x88AB;&#x8BA4;&#x4E3A;&#x662F;&#x6700;&#x7EC8;&#x6548;&#x5E94;&#x5668;&#x7AA6;&#x623F;&#x7ED3;&#x808C;&#x7EC6;&#x80DE;&#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x7684;&#x9635;&#x8425;&#x5F53;&#x4E2D;&#x3002;I(f) &#x88AB;&#x751F;&#x4EA7;&#x7684;&#x8D85;&#x6781;&#x5316;&#x6FC0;&#x6D3B;&#x73AF;&#x6838;&#x82F7;&#x9178;&#x654F;&#x611F; (HCN4) &#x901A;&#x9053;&#xFF0C;&#x8FD9;&#x53EF;&#x4EE5;&#x901A;&#x8FC7;&#x76F4;&#x63A5;&#x7ED1;&#x5B9A;&#x5230;&#x5B88;&#x6052;&#x73AF;&#x6838;&#x82F7;&#x9178;&#x7ED1;&#x5B9A;&#x57DF; C &#x603B;&#x7AD9;&#x7684;&#x6E20;&#x9053;&#x5728;&#x8425;&#x5730;&#x7684;&#x5F3A;&#x5316;&#x3002;beta I(f) &#x4E8E;&#x7AA6;&#x623F;&#x7ED3;&#x7684;&#x80BE;&#x4E0A;&#x817A;&#x7D20;&#x80FD;&#x8C03;&#x8282;&#x662F;&#x60F3;&#x901A;&#x8FC7;&#x8FD9;&#x79CD;&#x76F4;&#x63A5;&#x7ED1;&#x5B9A;&#x673A;&#x5236;&#xFF0C;&#x72EC;&#x7ACB;&#x78F7;&#x9178;&#x5316;&#x7684;&#x53D1;&#x751F;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x8C03;&#x67E5;&#x4E86;&#x662F;&#x5426;&#x8425;&#x6D3B;&#x5316;&#x86CB;&#x767D;&#x6FC0;&#x9176; (PKA) &#x8FD8;&#x53EF;&#x4EE5;&#x8C03;&#x8282;&#x7AA6;&#x623F;&#x7ED3; HCN4 &#x6E20;&#x9053;&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0; PKA &#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x5927;&#x5927;&#x51CF;&#x5C11;&#x4ECE;&#x5C0F;&#x9F20;&#x8F6C;&#x79FB; I(f) &#x7AA6;&#x623F;&#x7ED3;&#x808C;&#x7EC6;&#x80DE;&#x7684;&#x7535;&#x538B;&#x4F9D;&#x8D56;&#x6027; beta &#x80BE;&#x4E0A;&#x817A;&#x7D20;&#x80FD;&#x53D7;&#x4F53;&#x6FC0;&#x52A8;&#x5242;&#x7684;&#x80FD;&#x529B;&#x3002;PKA &#x4E5F;&#x8F6C;&#x79FB;&#x7ED9;&#x4E86;&#x8FD9;&#x4E24;&#x79CD;&#x57FA;&#x56E0;&#x8868;&#x8FBE; HCN4 &#x6E20;&#x9053;&#x66F4;&#x79EF;&#x6781;&#x6F5C;&#x529B;&#x7684;&#x6FC0;&#x6D3B;&#x7535;&#x538B;&#x4F9D;&#x8D56;&#x6027;&#x3002;&#x4F53;&#x5916;&#x78F7;&#x9178;&#x5316;&#x6D4B;&#x5B9A;&#x53CA;&#x8D28;&#x8C31;&#x6CD5;&#x900F;&#x9732; PKA &#x53EF;&#x4EE5;&#x76F4;&#x63A5; phosphorylate &#x81F3;&#x5C11; 13 &#x7F51;&#x7AD9; HCN4&#xFF0C;&#x5305;&#x62EC;&#x81F3;&#x5C11;&#x4E09;&#x4E2A;&#x6B8B;&#x7559;&#x5728; N &#x603B;&#x7AD9;&#x548C;&#x81F3;&#x5C11; 10 C &#x603B;&#x7AD9;&#x3002;&#x622A;&#x65AD;&#x548C;&#x4E19;&#x6C28;&#x9178;&#x53D6;&#x4EE3; HCN4 &#x6E20;&#x9053;&#x7684;&#x529F;&#x80FD;&#x5206;&#x6790;&#x786E;&#x5B9A; PKA &#x76D1;&#x7BA1;&#x7F51;&#x7AD9;&#x4E2D;&#x7684;&#x8FDC;&#x7AEF; C &#x603B;&#x7AD9;&#x7684; HCN4&#xFF0C;&#x8FD9;&#x662F; I(f) &#x7684; PKA &#x8C03;&#x5236;&#x7684;&#x8981;&#x6C42;&#x3002;&#x96C6;&#x4F53;&#xFF0C;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x663E;&#x793A;&#xFF0C;&#x672C;&#x673A;&#x548C; HCN4 &#x8868;&#x8FBE;&#x7684;&#x6E20;&#x9053;&#x53EF;&#x4EE5;&#x53D7; pka &#x503C;&#xFF0C;&#x5E76;&#x63D0;&#x9AD8;&#x8FD9;&#x4E00;&#x673A;&#x5236;&#x6709;&#x52A9;&#x4E8E;&#x5FC3;&#x7387;&#x7684;&#x4EA4;&#x611F;&#x795E;&#x7ECF;&#x8C03;&#x8282;&#x7684;&#x53EF;&#x80FD;&#x6027;&#x3002;

&#x78F7;&#x9178;&#x5316;&#x548C;&#x8D85;&#x6781;&#x5316;&#x6FC0;&#x6D3B;&#x86CB;&#x767D;&#x6FC0;&#x9176; A &#x5728;&#x9F20;&#x6807;&#x7AA6;&#x623F;&#x7ED3; HCN4 &#x9891;&#x9053;&#x7684;&#x8C03;&#x5236;&#x3002;

The sympathetic nervous system increases heart rate by activating beta adrenergic receptors and increasing cAMP levels in myocytes in the sinoatrial node. The molecular basis for this response is not well understood; however, the cardiac funny current (I(f)) is thought to be among the end effectors for cAMP signaling in sinoatrial myocytes. I(f) is produced by hyperpolarization-activated cyclic nucleotide-sensitive (HCN4) channels, which can be potentiated by direct binding of cAMP to a conserved cyclic nucleotide binding domain in the C terminus of the channels. beta Adrenergic regulation of I(f) in the sinoatrial node is thought to occur via this direct binding mechanism, independent of phosphorylation. Here, we have investigated whether the cAMP-activated protein kinase (PKA) can also regulate sinoatrial HCN4 channels. We found that inhibition of PKA significantly reduced the ability of beta adrenergic agonists to shift the voltage dependence of I(f) in isolated sinoatrial myocytes from mice. PKA also shifted the voltage dependence of activation to more positive potentials for heterologously expressed HCN4 channels. In vitro phosphorylation assays and mass spectrometry revealed that PKA can directly phosphorylate at least 13 sites on HCN4, including at least three residues in the N terminus and at least 10 in the C terminus. Functional analysis of truncated and alanine-substituted HCN4 channels identified a PKA regulatory site in the distal C terminus of HCN4, which is required for PKA modulation of I(f). Collectively, these data show that native and expressed HCN4 channels can be regulated by PKA, and raise the possibility that this mechanism could contribute to sympathetic regulation of heart rate.

Phosphorylation and modulation of hyperpolarization-activated HCN4 channels by protein kinase A in the mouse sinoatrial node.

Le syst&egrave;me nerveux sympathique augmente la fr&eacute;quence cardiaque en activant les r&eacute;cepteurs b&ecirc;ta adr&eacute;nergiques et en augmentant les taux d&#39;AMPc dans les myocytes dans le n&oelig;ud sino-auriculaire. La base mol&eacute;culaire de cette r&eacute;ponse n&#39;est pas bien comprise ; Cependant, le courant dr&ocirc;le cardiaque (concentrera est pens&eacute; pour &ecirc;tre parmi les effecteurs pour le cAMP de signalisation dans les myocytes sino-auriculaire. On&eacute;reuses est produit par activ&eacute;s par une hyperpolarisation cyclique nucl&eacute;otides (HCN4) canaux sensibles, qui peut &ecirc;tre potentialis&eacute;s par liaison directe du cAMP pour un domaine de liaison de nucl&eacute;otides cycliques conserv&eacute;e dans la partie C terminale des canaux. b&ecirc;ta adr&eacute;nergique r&egrave;glement d&#39;on&eacute;reuses dans le n&oelig;ud sinusal est pens&eacute; pour se produire par l&#39;interm&eacute;diaire de ce m&eacute;canisme de liaison directe, ind&eacute;pendant de la phosphorylation. Ici, nous avons examin&eacute; si la cAMP-activated protein kinase (PKA) peut &eacute;galement r&eacute;glementer sino-auriculaire HCN4 canaux. Nous avons trouv&eacute; que l&#39;inhibition de la PKA a consid&eacute;rablement r&eacute;duit la capacit&eacute; des substances b&eacute;ta-agonistes adr&eacute;nergiques de passer de la d&eacute;pendance de la tension d&#39;on&eacute;reuses dans des myocytes isol&eacute;s sino-auriculaire de souris. PKA d&eacute;cal&eacute; aussi la d&eacute;pendance de la tension d&#39;activation &agrave; des potentiels plus positifs pour les canaux de HCN4 fa&ccedil;on h&eacute;t&eacute;rologue exprim&eacute;e. Des essais in vitro de la phosphorylation et la spectrom&eacute;trie de masse a r&eacute;v&eacute;l&eacute; que le PKA peut phosphoryler directement au moins 13 sites sur HCN4, y compris au moins trois r&eacute;sidus &agrave; l&#39;extr&eacute;mit&eacute; N terminale et au moins 10 dans la partie C terminale. Analyse fonctionnelle des canaux de HCN4 tronqu&eacute; et alanine-substitu&eacute;s identifi&eacute; un site r&eacute;gulateur PKA dans le terminus de C distal de HCN4, qui est requis pour la modulation de la PKA d&#39;on&eacute;reuses. Collectivement, ces donn&eacute;es montrent que les indig&egrave;nes et exprim&eacute; HCN4 canaux peut &ecirc;tre r&eacute;gl&eacute;e par la PKA et soul&egrave;vent la possibilit&eacute; que ce m&eacute;canisme pourrait contribuer au r&egrave;glement sympathique de la fr&eacute;quence cardiaque.

La phosphorylation et la modulation des canaux HCN4 une hyperpolarisation de la prot&eacute;ine kinase A, dans le n&oelig;ud sino-auriculaire de souris.

Der Sympathikus erh&ouml;ht Herzfrequenz um Beta-adrenerge Rezeptoren aktivieren und cAMP Level in Muskelzellen in den Sinoatrial-Knoten. Die molekulare Grundlage f&uuml;r diese Antwort ist nicht gut verstanden; jedoch die kardiale lustige aktuelle (I(f)) ist vermutlich unter der Endeffektoren f&uuml;r cAMP signalisieren in den Schrittmacherzellen Myocytes. I(f) ist durch Hyperpolarisation aktiviert zyklische Nukleotid-Sensitive (HCN4) Kan&auml;le hergestellt, die durch direkte Bindung des Lagers einer konservierten zyklische Nukleotid-Bindung-Dom&auml;ne in der Carboxyl-Terminus der Kan&auml;le potenzierte werden k&ouml;nnen. Beta-adrenerge Regulierung der I(f) in den Sinoatrial-Knoten ist gedacht, um &uuml;ber diese direkte Bindungsmechanismus, unabh&auml;ngig von Phosphorylierung auftreten. Hier haben wir untersucht, ob die Lager-aktivierte Proteinkinase (PKA) auch Schrittmacherzellen HCN4 Kan&auml;le regulieren kann. Wir fanden, dass die Hemmung der PKA erheblich verringert die F&auml;higkeit der adrenergen Agonisten, die Abh&auml;ngigkeit der Spannung der I(f) in isolierten Schrittmacherzellen Myocytes von M&auml;usen zu verlagern. PKA verlagerte auch die Abh&auml;ngigkeit der Spannung der Aktivierung auf positivere Potenziale f&uuml;r heterologously exprimierten HCN4 Kan&auml;le. In-vitro-Phosphorylierung-Assays und Massenspektrometrie ergab, dass mindestens 13 Orte auf HCN4, einschlie&szlig;lich mindestens drei R&uuml;ckst&auml;nde in der N-Terminus und mindestens 10 in das C-Terminus direkt von PKA phosphoryliert kann. Funktionsanalyse der abgeschnittene und Alanin-substituierte HCN4 Kan&auml;le identifiziert eine regulatorische PKA-Website in das distale C-Terminus von HCN4, die f&uuml;r PKA Modulation der I(f) erforderlich ist. Zusammen diese Daten zeigen, dass native und exprimierten HCN4 Kan&auml;le von PKA reguliert werden k&ouml;nnen und die M&ouml;glichkeit, die diesen Mechanismus zu sympathisch Regulierung der Herzfrequenz zu erh&ouml;hen.

Phosphorylierung und Modulation der Hyperpolarisation aktiviert HCN4-Channels von Proteinkinase A in den Maus-Sinoatrial-Knoten.

Il sistema nervoso simpatico aumenta la frequenza cardiaca attivando i recettori adrenergici beta e aumentando i livelli di campi in miociti nel nodo senoatriale. La base molecolare per questa risposta non &egrave; ben chiaro; Tuttavia, la corrente funny cardiaca (I(f)) &egrave; pensato per essere tra gli effettori di fine per il campo di segnalazione in miociti senoatriale. Domanda &egrave; prodotto da hyperpolarization-attivato ciclici nucleotide-sensibili (HCN4) canali, che possono essere potenziate da binding diretto dell&#39;accampamento a un dominio di legame conservata nucleotide ciclico in C terminale dei canali. Beta adrenergico regolamento della domanda nel nodo senoatriale &egrave; pensato per verificarsi tramite questo meccanismo di binding diretto, indipendente di fosforilazione. Qui, abbiamo studiato se la chinasi di proteina accampamento-attivato (PKA) pu&ograve; anche regolare senoatriale HCN4 canali. Abbiamo trovato che l&#39;inibizione della PKA ridotto significativamente la capacit&agrave; di spostare la dipendenza di tensione dell&#39;acquisita in miociti senoatriale isolate da topi beta adrenergici. PKA spostata anche la dipendenza della tensione di attivazione a potenziali pi&ugrave; positivi per canali HCN4 heterologously espressi. Saggi in vitro fosforilazione e spettrometria di massa ha rivelato che la PKA direttamente pu&ograve; fosforilare almeno 13 siti su HCN4, tra cui almeno tre residui di capolinea N e almeno 10 in C terminale. Analisi funzionale dei canali HCN4 troncati e alanina-sostituiti identificato un sito normativo PKA in C terminale distale dell&#39;HCN4, che &egrave; necessaria per la modulazione di PKA della domanda. Collettivamente, questi dati mostrano che nativo ed espressi HCN4 canali possono essere regolamentate da PKA e aumentare la possibilit&agrave; che questo meccanismo potrebbe contribuire al simpatico regolazione della frequenza cardiaca.

Fosforilazione e modulazione di hyperpolarization-attivato HCN4 canali di chinasi di proteina A, nel nodo senoatriale del mouse.

&#x4EA4;&#x611F;&#x795E;&#x7D4C;&#x7CFB;&#x306F;&#x30D9;&#x30FC;&#x30BF; &#x30A2;&#x30C9;&#x30EC;&#x30CA;&#x30EA;&#x30F3;&#x53D7;&#x5BB9;&#x4F53;&#x3092;&#x6D3B;&#x6027;&#x5316;&#x3001;&#x514E;&#x6D1E;&#x3067;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B; cAMP &#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3092;&#x9AD8;&#x3081;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x5FC3;&#x62CD;&#x6570;&#x3092;&#x5897;&#x52A0;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x5FDC;&#x7B54;&#x306E;&#x5206;&#x5B50;&#x57FA;&#x76E4;&#x306F;&#x3088;&#x304F;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;;&#x305F;&#x3060;&#x3057;&#x3001;&#x73FE;&#x5728;&#x9762;&#x767D;&#x3044;&#x306E;&#x5FC3;&#x81D3; &#xFF08;I(f)) &#x3067;&#x3059;&#x7D42;&#x308F;&#x308A;&#x30A8;&#x30D5;&#x30A7;&#x30AF;&#x30BF;&#x306E;&#x6D1E;&#x623F;&#x7B4B;&#x7D30;&#x80DE;&#x5185;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x306E;&#x30AD;&#x30E3;&#x30F3;&#x30D7;&#x306E;&#x9593;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3068;&#x601D;&#x3063;&#x305F;&#x3002;I(f) &#x306E;&#x30AD;&#x30E3;&#x30F3;&#x30D7;&#x306B;&#x7BC0;&#x7D04;&#x3055;&#x308C;&#x305F;&#x74B0;&#x72B6;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x7D50;&#x5408;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x306E; C &#x672B;&#x7AEF;&#x3067;&#x306E;&#x76F4;&#x63A5;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x306B;&#x3088;&#x3063;&#x3066;&#x5897;&#x5F37;&#x306B;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x904E;&#x5206;&#x6975;&#x6D3B;&#x6027;&#x74B0;&#x72B6;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x611F;&#x53D7;&#x6027; (HCN4) &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306B;&#x3088;&#x3063;&#x3066;&#x751F;&#x6210;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x30D9;&#x30FC;&#x30BF; I(f) &#x514E;&#x6D1E;&#x306B;&#x304A;&#x3051;&#x308B;&#x30A2;&#x30C9;&#x30EC;&#x30CA;&#x30EA;&#x30F3;&#x306E;&#x898F;&#x5236;&#x306F;&#x3001;&#x3053;&#x306E;&#x76F4;&#x63A5;&#x30D0;&#x30A4;&#x30F3;&#x30C9; &#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x3001;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x306E;&#x72EC;&#x7ACB;&#x304C;&#x767A;&#x751F;&#x3059;&#x308B;&#x3068;&#x8003;&#x3048;&#x3089;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x6211;&#x3005; &#x306F;&#x30AD;&#x30E3;&#x30F3;&#x30D7;&#x6D3B;&#x6027;&#x5316;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30AD;&#x30CA;&#x30FC;&#x30BC; (PKA) &#x307E;&#x305F;&#x6D1E;&#x623F; HCN4 &#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x8ABF;&#x3079;&#x305F;&#x3002;&#x6211;&#x3005; &#x306F;&#x3001;&#x30DE;&#x30A6;&#x30B9;&#x304B;&#x3089;&#x5206;&#x96E2;&#x6D1E;&#x623F; myocytes &#x306E;&#x96FB;&#x5727;&#x4F9D;&#x5B58;&#x6027; I(f) &#x3092;&#x30B7;&#x30D5;&#x30C8;&#x3059;&#x308B;&#x3068;&#x3057;&#x3066;&#x3044;&#x308B;&#x30D9;&#x30FC;&#x30BF; &#x30A2;&#x30C9;&#x30EC;&#x30CA;&#x30EA;&#x30F3;&#x4F5C;&#x52D5;&#x85AC;&#x306E;&#x6FC0;&#x6E1B; PKA &#x306E;&#x963B;&#x5BB3;&#x3092;&#x767A;&#x898B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;PKA &#x307E;&#x305F;&#x96FB;&#x5727;&#x4F9D;&#x5B58;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D9;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306E; heterologously &#x8868;&#x73FE;&#x306E; HCN4 &#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x306E;&#x3088;&#x308A;&#x6B63;&#x96FB;&#x4F4D;&#x30B7;&#x30D5;&#x30C8;&#x3002;In vitro &#x3067;&#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x304A;&#x3088;&#x3073;&#x8CEA;&#x91CF;&#x5206;&#x6790; PKA &#x3092;&#x76F4;&#x63A5; HCN4&#x3001;&#x4E0A;&#x306E;&#x5C11;&#x306A;&#x304F;&#x3068;&#x3082; 13 &#x30B5;&#x30A4;&#x30C8;&#x306E; N &#x672B;&#x7AEF;&#x3068; C &#x672B;&#x7AEF;&#x306B; 10 &#x5C11;&#x306A;&#x304F;&#x3068;&#x3082;&#x3067;&#x5C11;&#x306A;&#x304F;&#x3068;&#x3082; 3 &#x6B8B;&#x57FA;&#x3092;&#x542B;&#x3080;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;PKA &#x898F;&#x5236;&#x30B5;&#x30A4;&#x30C8; HCN4 &#x306E;&#x9060;&#x4F4D; C &#x672B;&#x7AEF;&#x3067;&#x306E;&#x5207;&#x308A;&#x6368;&#x3066;&#x3068;&#x30A2;&#x30E9;&#x30CB;&#x30F3;&#x7F6E;&#x63DB; HCN4 &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x6A5F;&#x80FD;&#x89E3;&#x6790;&#x3092;&#x8B58;&#x5225;&#x3059;&#x308B;&#x5834;&#x5408;&#x306F; I(f) &#x306E; PKA &#x5909;&#x8ABF;&#x65B9;&#x5F0F;&#x304C;&#x5FC5;&#x8981;&#x306A;&#x3002;&#x7DCF;&#x79F0;&#x3057;&#x3066;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x8868;&#x793A;&#x3068;&#x8868;&#x73FE;&#x306E; HCN4 &#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB; PKA &#x306B;&#x3088;&#x3063;&#x3066;&#x898F;&#x5236;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3001;&#x3053;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306F;&#x5FC3;&#x62CD;&#x6570;&#x306E;&#x4EA4;&#x611F;&#x795E;&#x7D4C;&#x8ABF;&#x7BC0;&#x306B;&#x8CA2;&#x732E;&#x3067;&#x304D;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x9AD8;&#x3081;&#x307E;&#x3059;&#x3002;

&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3068;&#x5909;&#x8ABF;&#x306B;&#x3088;&#x308B;&#x30D7;&#x30ED;&#x30C6;&#x30A4;&#x30F3;&#x30AD;&#x30CA;&#x30FC;&#x30BC; A &#x30DE;&#x30A6;&#x30B9;&#x306E;&#x7D50;&#x7BC0;&#x5185;&#x306E; HCN4 &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x904E;&#x5206;&#x6975;&#x6D3B;&#x6027;&#x5316;&#x3057;&#x307E;&#x3059;&#x3002;

&#xAD50;&#xAC10; &#xC2E0; &#xACBD;&#xACC4;&#xB294; &#xBCA0;&#xD0C0; &#xC544;&#xB4DC;&#xB808;&#xB0A0;&#xB9B0; &#xC218;&#xC6A9; &#xCCB4;&#xB97C; &#xD65C;&#xC131;&#xD654; &#xD558; &#xACE0; sinoatrial &#xB178;&#xB4DC;&#xC5D0; myocytes&#xC5D0;&#xC11C; &#xCEA0;&#xD504; &#xB808;&#xBCA8;&#xC744; &#xC99D;&#xAC00; &#xD558; &#xC5EC; &#xC2EC;&#xC7A5; &#xBC15;&#xB3D9;&#xC744; &#xC99D;&#xAC00; &#xC2DC;&#xD0B5;&#xB2C8;&#xB2E4;. &#xC774; &#xC751;&#xB2F5;&#xC5D0; &#xB300; &#xD55C; &#xBD84;&#xC790; &#xAE30;&#xCD08;&#xB294; &#xC798; &#xC774;&#xD574; &#xB418;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;; &#xADF8;&#xB7EC;&#xB098; &#xC2EC;&#xC7A5; &#xC7AC; &#xBC0C; &#xC804;&#xB958; (I(f)) &#xCEA0;&#xD504; sinoatrial myocytes&#xC5D0;&#xC11C; &#xC2E0;&#xD638;&#xC5D0; &#xB300; &#xD55C; &#xC5D4;&#xB4DC; &#xC774;&#xD399;&#xD130; &#xB4E4; &#xAC83;&#xC73C;&#xB85C; &#xC0DD;&#xAC01; &#xB429;&#xB2C8;&#xB2E4;. I(f)&#xC5D0; &#xC758;&#xD574; hyperpolarization &#xD65C;&#xC131;&#xD654; &#xC8FC;&#xAE30;&#xC801; &#xB274;&#xD074;&#xB808;&#xC624;&#xD2F0;&#xB4DC; &#xAD6C;&#xBD84; (HCN4) &#xCC44;&#xB110;, &#xCC44;&#xB110;&#xC758; C &#xB9D0;&#xB2E8;&#xC5D0; &#xBCF4;&#xC874;&#xB41C; &#xC8FC;&#xAE30;&#xC801; &#xB274;&#xD074;&#xB808;&#xC624;&#xD2F0;&#xB4DC; &#xBC14;&#xC778;&#xB529; &#xB3C4;&#xBA54;&#xC778; &#xCEA0;&#xD504;&#xC758; &#xC9C1;&#xC811; &#xBC14;&#xC778;&#xB529;&#xC5D0;&#xC11C; potentiated &#xC218; &#xC788;&#xB294; &#xC0DD;&#xC0B0;. &#xBCA0;&#xD0C0; &#xC544;&#xB4DC;&#xB808;&#xB0A0;&#xB9B0; &#xADDC;&#xC815; i(f)&#xC758; sinoatrial &#xB178;&#xB4DC;&#xB294; &#xC778; &#xC0B0;&#xD654;&#xC758; &#xB3C5;&#xB9BD;&#xC774; &#xC9C1;&#xC811; &#xBC14;&#xC778;&#xB529; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xD1B5;&#xD574; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xAC83; &#xC0DD;&#xAC01; &#xB41C;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC; &#xCEA0;&#xD504; &#xD65C;&#xC131;&#xD654; &#xD55C; &#xB2E8;&#xBC31;&#xC9C8; &#xD0A4; &#xB2C8; &#xC544; &#xC81C; (PKA) &#xB610;&#xD55C; sinoatrial HCN4 &#xCC44;&#xB110;&#xC744; &#xC870;&#xC808; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC5EC;&#xBD80;&#xB97C; &#xC870;&#xC0AC; &#xD569;&#xB2C8;&#xB2E4;. PKA&#xC758; &#xC800;&#xD574; &#xD06C;&#xAC8C; &#xC950;&#xC5D0;&#xC11C; &#xACA9;&#xB9AC; &#xB41C; sinoatrial myocytes&#xC5D0;&#xC11C; i(f)&#xC758; &#xC804;&#xC555; &#xC758;&#xC874;&#xC131;&#xC744; &#xC774;&#xB3D9; &#xD558;&#xB294; &#xBCA0;&#xD0C0; &#xC544;&#xB4DC;&#xB808;&#xB0A0;&#xB9B0; agonists&#xC758; &#xAE30;&#xB2A5; &#xAC10;&#xC18C;&#xB97C; &#xCC3E;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4;. PKA&#xB294; &#xB610;&#xD55C; heterologously &#xD45C;&#xD604;&#xB41C; HCN4 &#xCC44;&#xB110;&#xC5D0; &#xB300; &#xD55C; &#xB354; &#xB9CE;&#xC740; &#xAE0D;&#xC815;&#xC801;&#xC778; &#xC7A0;&#xC7AC;&#xB825;&#xC744; &#xD65C;&#xC131;&#xD654;&#xC758; &#xC804;&#xC555; &#xC758;&#xC874;&#xC131;&#xC744; &#xC774;&#xB3D9;&#xD569;&#xB2C8;&#xB2E4;. &#xCCB4; &#xC678; &#xC778; &#xC0B0;&#xD654; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xACFC; &#xC9C8;&#xB7C9; &#xBD84;&#xC11D; &#xACF5;&#xAC1C; PKA N &#xB9D0;&#xB2E8;&#xC5D0;&#xC11C; &#xC801;&#xC5B4;&#xB3C4; 10 C &#xC885;&#xC810;&#xC5D0;&#xC11C; &#xC801;&#xC5B4;&#xB3C4; &#xC138; &#xAC00;&#xC9C0; &#xC794;&#xB958;&#xBB3C;&#xC744; &#xD3EC;&#xD568; &#xD558; &#xC5EC; HCN4&#xC5D0; &#xC801;&#xC5B4;&#xB3C4; 13 &#xC0AC;&#xC774;&#xD2B8; phosphorylate &#xC9C1;&#xC811; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC798;&#xB9B0;&#xACFC; &#xC54C;&#xB77C;&#xB2CC; &#xB300;&#xCCB4; HCN4 &#xCC44;&#xB110;&#xC758; &#xAE30;&#xB2A5; &#xBD84;&#xC11D; i(f)&#xC758; PKA &#xBCC0;&#xC870;&#xC5D0; &#xD544;&#xC694;&#xD55C; HCN4&#xC758; &#xC6D0;&#xC2EC; C &#xC885;&#xC810;&#xC5D0;&#xC11C; PKA &#xADDC;&#xC81C; &#xC0AC;&#xC774;&#xD2B8; &#xBC1C;&#xACAC;. &#xC0E8; &#xB2E4;, &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130;&#xB97C; &#xB124;&#xC774;&#xD2F0;&#xBE0C; &#xD45C;&#xC2DC; &#xBC0F; &#xD45C;&#xD604;&#xB41C; HCN4 &#xCC44;&#xB110; PKA, &#xADDC;&#xC81C; &#xC218; &#xADF8;&#xB9AC;&#xACE0;&#xC774; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC740; &#xC2EC;&#xC7A5; &#xBC15;&#xB3D9;&#xC758; &#xACF5;&#xAC10; &#xADDC;&#xC81C;&#xC5D0; &#xAE30;&#xC5EC;&#xD560; &#xC218; &#xC788;&#xB294; &#xAC00;&#xB2A5;&#xC131;&#xC744; &#xC81C;&#xAE30;.

&#xC778; &#xC0B0;&#xD654; &#xBC0F; &#xB2E8;&#xBC31;&#xC9C8; &#xD0A4; &#xB2C8; &#xC544; &#xC81C; A &#xB9C8;&#xC6B0;&#xC2A4; sinoatrial &#xB178;&#xB4DC;&#xC5D0; &#xC758;&#xD574; hyperpolarization &#xD65C;&#xC131;&#xD654; HCN4 &#xCC44;&#xB110;&#xC758; &#xBCC0;&#xC870;.

El sistema nervioso simp&aacute;tico aumenta la frecuencia card&iacute;aca activando receptores adren&eacute;rgicos beta y aumentando los niveles de campo en miocitos en el n&oacute;dulo sinoauricular. La base molecular para esta respuesta no es bien conocida; sin embargo, la corriente graciosa cardiaca (I(f)) se cree que entre los efectores finales para campamento en miocitos sinoauricular. I(f) es producido por hiperpolarizaci&oacute;n c&iacute;clicos sensibles al nucle&oacute;tido (HCN4) canales activados, que pueden ser potenciados por atascamiento directo del campo a un dominio de uni&oacute;n de nucle&oacute;tido c&iacute;clico conservado en la terminal C de los canales. beta adren&eacute;rgico regulaci&oacute;n de I(f) en el n&oacute;dulo sinoauricular se piensa para ocurrir a trav&eacute;s de este mecanismo de enlace directo, independiente de la fosforilaci&oacute;n. Aqu&iacute;, hemos investigado si el campamento quinasa activada (PKA) tambi&eacute;n puede regular sinoauricular HCN4 canales. Encontramos que la inhibici&oacute;n de PKA redujo significativamente la capacidad de los agonistas adren&eacute;rgicos beta para cambiar la dependencia de voltaje de I(f) en miocitos sinoauricular aislados de ratones. PKA cambi&oacute; tambi&eacute;n la dependencia de voltaje de activaci&oacute;n a potenciales m&aacute;s positivos para los canales de HCN4 heterologously expresados. Ensayos in vitro de la fosforilaci&oacute;n y espectrometr&iacute;a de masas revelaron que el PKA puede fosforilan directamente por lo menos 13 sitios en HCN4, incluyendo al menos tres residuos en la terminal N y por lo menos 10 en la terminal C. An&aacute;lisis funcional de truncado y alanina-substituted HCN4 canales identificado un sitio reglamentario de PKA en la terminal C distal del HCN4, que se requiere para la modulaci&oacute;n de la PKA de I(f). En conjunto, estos datos demuestran nativos y expresados HCN4 canales pueden ser regulados por PKA y aumentar la posibilidad de que este mecanismo podr&iacute;a contribuir a la regulaci&oacute;n comprensiva del ritmo card&iacute;aco.

Fosforilaci&oacute;n y modulaci&oacute;n de canales de HCN4 activada por hiperpolarizaci&oacute;n por kinase de prote&iacute;na A en el rat&oacute;n n&oacute;dulo sinoauricular.

On pense que le courant dr&ocirc;le, on&eacute;reuses, dans les myocytes sino-auriculaire contribuent &agrave; l&#39;augmentation de lutte ou de fuite sympathique dans la fr&eacute;quence cardiaque. On&eacute;reuses est produite par les cha&icirc;nes de nucl&eacute;otides cycliques activ&eacute;s par une hyperpolarisation sensibles-4 (HCN4), et il est largement admis que sympathique r&egrave;glement d&#39;on&eacute;reuses s&#39;effectue via une liaison directe du cAMP &agrave; HCN4, ind&eacute;pendant de la phosphorylation. Cependant, nous avons r&eacute;cemment montr&eacute; que l&#39;activit&eacute; prot&eacute;ine Kinase A (PKA) est requise pour r&egrave;glement sympathique d&#39;on&eacute;reuses et que le PKA peut phosphoryler directement HCN4. Dans la pr&eacute;sente &eacute;tude, nous avons examin&eacute; les effets d&#39;un peptide inhibiteur de la PKA de myristoylated (myr-PKI) sur i dans les myocytes sino-auriculaire de souris. Nous avons trouv&eacute; que myr-infrastructure &agrave; cl&eacute; publique et un autre peptide myristoylated puissamment et sp&eacute;cifiquement potentialisent on&eacute;reuses via un m&eacute;canisme qui n&#39;impliquaient pas d&#39;inhibition de la PKA et &eacute;tait ind&eacute;pendante de la s&eacute;quence peptidique, prot&eacute;ine Kinase C ou phosphatidylinositol-4, 5-bisphosphate. L&#39;activation hors cible d&#39;on&eacute;reuses de peptides myristoylated limite leur utilit&eacute; pour l&#39;&eacute;tude des m&eacute;canismes de stimulateur cardiaque dans les myocytes sino-auriculaire.

Les peptides myristoylated potentialisent le courant dr&ocirc;le (concentrera dans les myocytes sino-auriculaire.

&#x6ED1;&#x7A3D;&#x7684;&#x7535;&#x6D41; I(f)&#xFF0C;&#x7AA6;&#x623F;&#x7ED3;&#x808C;&#x7EC6;&#x80DE;&#x88AB;&#x8BA4;&#x4E3A;&#x6709;&#x52A9;&#x4E8E;&#x4EA4;&#x611F;&#x795E;&#x7ECF;&#x7684;&#x6597;&#x4E89;&#x6216;&#x98DE;&#x884C;&#x589E;&#x52A0;&#x5FC3;&#x7387;&#x3002;I(f) &#x7531;&#x8D85;&#x6781;&#x5316;&#x6FC0;&#x6D3B;&#x73AF;&#x6838;&#x82F7;&#x9178;&#x654F;&#x611F; 4 (HCN4) &#x6E20;&#x9053;&#xFF0C;&#x548C;&#x4EBA;&#x4EEC;&#x666E;&#x904D;&#x8BA4;&#x4E3A;&#x7684; I(f) &#x7684;&#x4EA4;&#x611F;&#x795E;&#x7ECF;&#x8C03;&#x8282;&#x53D1;&#x751F;&#x901A;&#x8FC7;&#x76F4;&#x63A5;&#x7ED1;&#x5B9A;&#x5230; HCN4&#xFF0C;&#x78F7;&#x9178;&#x5316;&#x7684;&#x72EC;&#x7ACB;&#x8425;&#x3002;&#x4E0D;&#x8FC7;&#xFF0C;&#x6211;&#x4EEC;&#x6700;&#x8FD1;&#x8868;&#x660E;&#x86CB;&#x767D;&#x6FC0;&#x9176; A &#xFF08;PKA&#xFF09; &#x6D3B;&#x52A8;&#x662F; I(f) &#x7684;&#x4EA4;&#x611F;&#x795E;&#x7ECF;&#x76D1;&#x7BA1;&#x6240;&#x5FC5;&#x9700;&#x7684;&#x548C; PKA &#x53EF;&#x4EE5;&#x76F4;&#x63A5; phosphorylate HCN4&#x3002;&#x5728;&#x672C;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x6211;&#x4EEC;&#x7814;&#x7A76; myristoylated PKA &#x6291;&#x5236;&#x80BD; (&#x9A6C;&#x5E01; PKI) &#x5F71;&#x54CD;&#x7684; I(f) &#x9F20;&#x6807;&#x7AA6;&#x623F;&#x7ED3;&#x808C;&#x7EC6;&#x80DE;&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x9A6C;&#x5E01; PKI &#x548C;&#x53E6;&#x4E00;&#x4E2A; myristoylated &#x80BD;&#x5148;&#x5929;&#x548C;&#x4E13;&#x95E8;&#x5F3A;&#x5316; I(f) &#x901A;&#x8FC7;&#x4E00;&#x79CD;&#x673A;&#x5236;&#xFF0C;&#x5E76;&#x4E0D;&#x6D89;&#x53CA; PKA &#x6291;&#x5236;&#x4F5C;&#x7528;&#xFF0C;&#x8FD9;&#x5C31;&#x662F;&#x72EC;&#x7ACB;&#x7684;&#x591A;&#x80BD;&#x5E8F;&#x5217;&#x3001; &#x86CB;&#x767D;&#x6FC0;&#x9176; C &#x6216;&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187;-4&#xFF0C;5-&#x4E8C;&#x78F7;&#x9178;&#x3002;I(f) myristoylated &#x591A;&#x80BD;&#x7684;&#x504F;&#x79BB;&#x76EE;&#x6807;&#x6FC0;&#x6D3B;&#x9650;&#x5236;&#x5176;&#x6548;&#x7528;&#x7684;&#x808C;&#x7EC6;&#x80DE;&#x7AA6;&#x623F;&#x7ED3;&#x8D77;&#x640F;&#x5668;&#x673A;&#x5236;&#x7684;&#x7814;&#x7A76;&#x3002;

Myristoylated &#x591A;&#x80BD;&#x4FC3;&#x8FDB;&#x6ED1;&#x7A3D;&#x7684;&#x7535;&#x6D41; (I(f)) &#x7AA6;&#x623F;&#x7ED3;&#x808C;&#x7EC6;&#x80DE;&#x3002;

The funny current, I(f), in sinoatrial myocytes is thought to contribute to the sympathetic fight-or-flight increase in heart rate. I(f) is produced by hyperpolarization-activated cyclic nucleotide sensitive-4 (HCN4) channels, and it is widely believed that sympathetic regulation of I(f) occurs via direct binding of cAMP to HCN4, independent of phosphorylation. However, we have recently shown that Protein Kinase A (PKA) activity is required for sympathetic regulation of I(f) and that PKA can directly phosphorylate HCN4. In the present study, we examined the effects of a myristoylated PKA inhibitory peptide (myr-PKI) on I(f) in mouse sinoatrial myocytes. We found that myr-PKI and another myristoylated peptide potently and specifically potentiated I(f) via a mechanism that did not involve PKA inhibition and that was independent of the peptide sequence, Protein Kinase C, or phosphatidylinositol-4,5-bisphosphate. The off-target activation of I(f) by myristoylated peptides limits their usefulness for studies of pacemaker mechanisms in sinoatrial myocytes.

Myristoylated peptides potentiate the funny current (I(f)) in sinoatrial myocytes.

Die lustige aktuelle I(f), in den Schrittmacherzellen Myocytes ist vermutlich der sympathische K&auml;mpfen-oderflug Anstieg der Herzfrequenz beitragen. I(f) wird durch Hyperpolarisation aktiviert zyklisches Nukleotid Sensitive-4 (HCN4) Kan&auml;le produziert, und es wird angenommen, dass sympathische Regulierung der I(f) &uuml;ber direkte Bindung des Lagers zu HCN4, unabh&auml;ngig von Phosphorylierung tritt. Jedoch haben wir vor kurzem gezeigt, dass Protein Kinase A (PKA) Aktivit&auml;t f&uuml;r die sympathische Regulierung der I(f) erforderlich ist und dass PKA HCN4 direkt phosphoryliert kann. In der vorliegenden Studie untersuchten wir die Wirkung von Myristoylated PKA hemmenden Peptid (Myr-PKI) auf I(f) in den Schrittmacherzellen Myocytes Maus. Wir fanden, dass Myr-PKI und anderen Myristoylated Peptid dabei wirksam und gezielt I(f) &uuml;ber einen Mechanismus potenzierte, die PKA Hemmung nicht beinhalten, und zwar unabh&auml;ngig von der Sequenz Peptid, Protein-Kinase-C oder Phosphatidylinositol-4, 5-bisphosphat. Die Weg-Ziel-Aktivierung von I(f) von Myristoylated Peptiden ihre N&uuml;tzlichkeit f&uuml;r Studien der Schrittmacher Mechanismen in den Schrittmacherzellen Myocytes eingeschr&auml;nkt.

Myristoylated Peptide potenzieren die lustige aktuelle (I(f)) in den Schrittmacherzellen Myocytes.

La corrente funny, acquisita, in miociti senoatriale &egrave; pensata per contribuire al simpatico fight-or-flight aumento nella frequenza cardiaca. Domanda &egrave; prodotto da nucleotide ciclico hyperpolarization-attivato sensibili-4 (HCN4) canali, ed &egrave; opinione diffusa che simpatico regolazione della domanda avviene tramite binding diretto dell&#39;accampamento a HCN4, indipendente di fosforilazione. Tuttavia, abbiamo recentemente dimostrato che attivit&agrave; della proteina chinasi A (PKA) &egrave; obbligatorio per regolamento comprensivo di domanda e che PKA direttamente pu&ograve; fosforilare HCN4. Nel presente studio, abbiamo esaminato gli effetti di un peptide inibitorio (myr-PKI) PKA myristoylated su domanda in miociti senoatriale del mouse. Abbiamo trovato che myr-PKI e un altro peptide myristoylated potentemente e specificamente potenziate acquisita attraverso un meccanismo che non coinvolgono l&#39;inibizione PKA e che &egrave; stato indipendente della sequenza del peptide, proteina chinasi C o fosfatidilinositolo 4,5-bisfosfato. L&#39;attivazione fuori bersaglio di domanda di peptidi myristoylated limita la loro utilit&agrave; per gli studi dei meccanismi di pacemaker in miociti senoatriale.

Peptidi myristoylated potenziare la corrente funny (I(f)) in miociti senoatriale.

&#x304A;&#x304B;&#x3057;&#x3044;&#x96FB;&#x6D41;&#x3001;I(f)&#x3001;&#x6D1E;&#x623F;&#x7B4B;&#x4EA4;&#x611F;&#x795E;&#x7D4C;&#x306E;&#x6226;&#x3044;&#x307E;&#x305F;&#x306F;&#x98DB;&#x884C;&#x5897;&#x52A0;&#x5FC3;&#x62CD;&#x6570;&#x306B;&#x8CA2;&#x732E;&#x3059;&#x308B;&#x3068;&#x8003;&#x3048;&#x3089;&#x308C;&#x307E;&#x3059;&#x3002;I(f) &#x904E;&#x5206;&#x6975;&#x6D3B;&#x6027;&#x74B0;&#x72B6;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x611F;&#x53D7;&#x6027; 4 (HCN4) &#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306B;&#x3088;&#x3063;&#x3066;&#x767A;&#x751F;&#x3057;&#x3001;I(f) &#x306E;&#x4EA4;&#x611F;&#x795E;&#x7D4C;&#x8ABF;&#x7BC0;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x30AD;&#x30E3;&#x30F3;&#x30D7; HCN4&#x3001;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x306E;&#x72EC;&#x7ACB;&#x3078;&#x306E;&#x76F4;&#x63A5;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x304C;&#x767A;&#x751F;&#x3059;&#x308B;&#x3053;&#x3068;&#x3001;&#x305D;&#x308C;&#x304C;&#x5E83;&#x304F;&#x4FE1;&#x3058;&#x3089;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x6211;&#x3005; &#x306F;&#x6700;&#x8FD1;&#x86CB;&#x767D;&#x8CEA;&#x30AD;&#x30CA;&#x30FC;&#x30BC; A(PKA) &#x6D3B;&#x52D5;&#x304C;&#x4EA4;&#x611F;&#x795E;&#x7D4C;&#x306E;&#x898F;&#x5236; I(f) &#x306E;&#x305F;&#x3081;&#x5FC5;&#x8981;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3068; PKA &#x3092;&#x76F4;&#x63A5; HCN4 &#x30EA;&#x30F3;&#x9178;&#x5316;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x69CB;&#x9020;&#x306E; PKA &#x963B;&#x5BB3;&#x30DA;&#x30D7;&#x30C1;&#x30C9; (myr PKI) &#x306E;&#x5F71;&#x97FF;&#x306B; I(f) &#x30DE;&#x30A6;&#x30B9;&#x6D1E;&#x623F; myocytes &#x3067;&#x691C;&#x8A0E;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005; &#x306F;&#x5897;&#x5F37; myr PKI &#x3068;&#x5225;&#x69CB;&#x9020;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x5F37;&#x529B;&#x3068;&#x5177;&#x4F53;&#x7684;&#x306B;&#x306F; I(f) &#x306F; PKA &#x6291;&#x5236;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x306A;&#x3044;&#x3068;&#x3055;&#x308C;&#x305F;&#x30DA;&#x30D7;&#x30C1;&#x30C3;&#x30C9; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3001;&#x86CB;&#x767D;&#x8CEA;&#x30AD;&#x30CA;&#x30FC;&#x30BC; C &#x307E;&#x305F;&#x306F;&#x30DB;&#x30B9;&#x30D5;&#x30A1;&#x30C1;&#x30B8;&#x30EB;&#x30A4;&#x30CE;&#x30B7;&#x30C8;&#x30FC;&#x30EB; 4, 5-&#x30D3;&#x30B9;&#x30EA;&#x30F3;&#x9178;&#x306E;&#x72EC;&#x7ACB;&#x3057;&#x305F;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x7D4C;&#x7531;&#x3057;&#x305F;&#x3053;&#x3068;&#x3092;&#x767A;&#x898B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;I(f) &#x69CB;&#x9020;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x306B;&#x3088;&#x308B;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8; &#x30AA;&#x30D5;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x306B;&#x304A;&#x3051;&#x308B;&#x6D1E;&#x623F; myocytes &#x3067;&#x30DA;&#x30FC;&#x30B9; &#x30E1;&#x30FC;&#x30AB;&#x30FC;&#x6A5F;&#x69CB;&#x306E;&#x6709;&#x7528;&#x6027;&#x304C;&#x5236;&#x9650;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;

&#x69CB;&#x9020;&#x30DA;&#x30D7;&#x30C1;&#x30C9;&#x5897;&#x5F37;&#x304A;&#x304B;&#x3057;&#x3044;&#x96FB;&#x6D41; (&#x6D1E;&#x623F; myocytes &#x3067; I(f))&#x3002;

Sinoatrial myocytes&#xC5D0;&#xC11C; &#xC7AC;&#xBBF8; &#xC788;&#xB294; &#xC804;&#xB958; I(f), &#xC2EC;&#xC7A5; &#xBC15;&#xB3D9;&#xC758; &#xACF5;&#xAC10; &#xC2F8;&#xC6C0; &#xB610;&#xB294; &#xBE44;&#xD589; &#xC99D;&#xAC00;&#xC5D0; &#xAE30;&#xC5EC;&#xD560; &#xC0DD;&#xAC01;&#xC785;&#xB2C8;&#xB2E4;. I(f) &#xC21C;&#xD658; &#xB274;&#xD074;&#xB808;&#xC624;&#xD2F0;&#xB4DC; hyperpolarization &#xD65C;&#xC131;&#xD654;&#xC5D0; &#xBBFC;&#xAC10;&#xD55C;-4 (HCN4) &#xCC44;&#xB110;&#xC5D0; &#xC758;&#xD574; &#xC0DD;&#xC0B0; &#xD558; &#xACE0; &#xB110;&#xB9AC; &#xACF5;&#xAC10; &#xADDC;&#xC815; i(f)&#xC758; HCN4, &#xC778; &#xC0B0;&#xD654;&#xC758; &#xB3C5;&#xB9BD;&#xC5D0; &#xCEA0;&#xD504;&#xC758; &#xC9C1;&#xC811; &#xBC14;&#xC778;&#xB529;&#xC744; &#xD1B5;&#xD574; &#xBC1C;&#xC0DD; &#xBBFF;&#xC5B4;&#xC9C4;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC6B0;&#xB9AC;&#xB294; &#xB2E8;&#xBC31;&#xC9C8; &#xD0A4; &#xB2C8; &#xC544; &#xC81C; A (PKA) &#xD65C;&#xB3D9; i(f)&#xC758; &#xACF5;&#xAC10; &#xADDC;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC694; &#xD558; &#xACE0; PKA HCN4 phosphorylate &#xC9C1;&#xC811; &#xC218; &#xD45C;&#xC2DC; &#xCD5C;&#xADFC;&#xC5D0;. &#xD604;&#xC7AC;&#xC758; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C;&#xB294; &#xC6B0;&#xB9AC; &#xB9C8;&#xC6B0;&#xC2A4; sinoatrial myocytes&#xC5D0;&#xC11C; i(f)&#xC5D0; myristoylated PKA &#xAE08;&#xC9C0; &#xD3A9;&#xD0C0;&#xC774;&#xB4DC; (myr PKI)&#xC758; &#xD6A8;&#xACFC; &#xAC80;&#xC0AC;&#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; myr PKI &#xBC0F; &#xB2E4;&#xB978; myristoylated &#xD3A9;&#xD0C0;&#xC774;&#xB4DC; potently &#xACE0; &#xD2B9;&#xBCC4;&#xD788; potentiated i(f)&#xB294; PKA &#xC5B5;&#xC81C;&#xB97C; &#xD3EC;&#xD568; &#xD558;&#xC9C0; &#xC54A;&#xC558;&#xB2E4; &#xD558; &#xACE0; &#xD3A9; &#xD2F0; &#xB4DC; &#xC21C;&#xC11C;, &#xB2E8;&#xBC31;&#xC9C8; &#xD0A4; &#xB2C8; &#xC544; &#xC81C; C, &#xB610;&#xB294; phosphatidylinositol-4, 5-bisphosphate&#xC758; &#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xD1B5;&#xD574; &#xBC1C;&#xACAC;. Myristoylated &#xD3A9; &#xD2F0; &#xB4DC;&#xC5D0; &#xC758;&#xD574; i(f)&#xC758; &#xB300;&#xC0C1;&#xC5D0;&#xC11C; &#xD65C;&#xC131;&#xD654; sinoatrial myocytes&#xC5D0;&#xC11C; &#xC2EC;&#xC7A5; &#xBC15; &#xB3D9;&#xAE30; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC758; &#xC5F0;&#xAD6C;&#xC5D0; &#xB300; &#xD55C; &#xADF8;&#xB4E4;&#xC758; &#xC720;&#xC6A9;&#xC131;&#xC744; &#xC81C;&#xD55C;&#xD569;&#xB2C8;&#xB2E4;.

&#xD3A9; &#xD2F0; &#xB4DC; Myristoylated &#xC57D;&#xD6A8; &#xC7AC; &#xBC0C; &#xC804;&#xB958; (I(f)) sinoatrial myocytes&#xC5D0;&#xC11C;.

La corriente graciosa, I(f), en miocitos sinoauricular se piensa contribuir al aumento de lucha o hu&iacute;da comprensivo en el ritmo card&iacute;aco. I(f) es producido por canales de nucle&oacute;tico c&iacute;clico activada por hiperpolarizaci&oacute;n sensible-4 (HCN4), y se cree ampliamente que regulaci&oacute;n comprensiva de I(f) se produce a trav&eacute;s de atascamiento directo del campo a HCN4, independiente de la fosforilaci&oacute;n. Sin embargo, recientemente hemos demostrado que la actividad de prote&iacute;na quinasa A (PKA) es necesario para regulaci&oacute;n comprensiva de I(f) y que el PKA puede fosforilan directamente HCN4. En el presente estudio, se examinaron los efectos de un p&eacute;ptido inhibitorio del PKA de myristoylated (myr-PKI) en I(f) en miocitos sinoauricular de rat&oacute;n. Encontramos que myr-PKI y otro p&eacute;ptido myristoylated potentemente y especialmente potenciada I(f) mediante un mecanismo que no implic&oacute; la inhibici&oacute;n de la PKA y que era independiente de la secuencia del p&eacute;ptido, prote&iacute;na quinasa C o Fosfatidilinositol-4, 5-bifosfato. La activaci&oacute;n de fuera del objetivo de I(f) por myristoylated p&eacute;ptidos limita su utilidad para los estudios de mecanismos de marcapasos en miocitos sinoauricular.

Myristoylated p&eacute;ptidos potencian la corriente graciosa (I(f)) en miocitos sinoauricular.

Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels produce the I(f) and I(h) currents, which are critical for cardiac pacemaking and neuronal excitability, respectively. HCN channels are modulated by cyclic AMP (cAMP), which binds to a conserved cyclic nucleotide-binding domain (CNBD) in the C terminus. The unliganded CNBD has been shown to inhibit voltage-dependent gating of HCNs, and cAMP binding relieves this "autoinhibition," causing a depolarizing shift in the voltage dependence of activation. Here we report that relief of autoinhibition can occur in the absence of cAMP in a cellular context- and isoform-dependent manner: when the HCN4 isoform was expressed in Chinese hamster ovary (CHO) cells, the basal voltage dependence was already shifted to more depolarized potentials and cAMP had no further effect on channel activation. This "pre-relief" of autoinhibition was specific both to HCN4 and to CHO cells; cAMP shifted the voltage dependence of HCN2 in CHO cells and of HCN4 in human embryonic kidney (HEK) cells. The pre-relief phenotype did not result from different concentrations of soluble intracellular factors in CHO and HEK cells, as it persisted in excised cell-free patches. Likewise, it did not arise from a failure of cAMP to bind to the CNBD of HCN4 in CHOs, as indicated by cAMP-dependent slowing of deactivation. Instead, a unique ∼300-amino acid region of the distal C terminus of HCN4 (residues 719-1012, downstream of the CNBD) was found to be necessary, but not sufficient, for the depolarized basal voltage dependence and cAMP insensitivity of HCN4 in CHO cells. Collectively, these data suggest a model in which multiple HCN4 channel domains conspire with membrane-associated intracellular factors in CHO cells to relieve autoinhibition in HCN4 channels in the absence of cAMP. These findings raise the possibility that such ligand-independent regulation could tune the activity of HCN channels and other CNBD-containing proteins in many physiological systems.

Cellular context and multiple channel domains determine cAMP sensitivity of HCN4 channels: ligand-independent relief of autoinhibition in HCN4.

Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN4) channels produce the "funny current," I(f), which contributes to spontaneous pacemaking in sinoatrial myocytes (SAMs). The C-terminus of HCN channels inhibits voltage-dependent gating, and cAMP binding relieves this "autoinhibition." We previously showed 1) that autoinhibition in HCN4 can be relieved in the absence of cAMP in some cellular contexts and 2) that PKA is required for β adrenergic receptor (βAR) signaling to HCN4 in SAMs. Together, these results raise the possibility that native HCN channels in SAMs may be insensitive to direct activation by cAMP. Here, we examined PKA-independent activation of If by cAMP in SAMs. We observed similar robust activation of If by exogenous cAMP and Rp-cAMP (an analog than cannot activate PKA). Thus PKA-dependent βAR-to-HCN signaling does not result from cAMP insensitivity of sinoatrial HCN channels and might instead arise via PKA-dependent limitation of cAMP production and/or cAMP access to HCN channels in SAMs.

PKA-independent activation of I(f) by cAMP in mouse sinoatrial myocytes.

The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by "funny" current (If) through HCN4 channels (the "Membrane Clock" hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two ("Coupled Clock"). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to β-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of If in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN.

Complete atrial-specific knockout of sodium-calcium exchange eliminates sinoatrial node pacemaker activity.

Pacemaker myocytes in the sinoatrial node of the heart initiate each heartbeat by firing spontaneous action potentials. However, the molecular processes that underlie pacemaking are incompletely understood, in part because of our limited ability to manipulate protein expression within the native cellular context of sinoatrial node myocytes (SAMs). Here we describe a new method for the culture of fully differentiated SAMs from adult mice, and we demonstrate that robust expression of introduced proteins can be achieved within 24-48 h in vitro via adenoviral gene transfer. Comparison of morphological and electrophysiological characteristics of 48 h-cultured versus acutely isolated SAMs revealed only minor changes in vitro. Specifically, we found that cells tended to flatten in culture but retained an overall normal morphology, with no significant changes in cellular dimensions or membrane capacitance. Cultured cells beat spontaneously and, in patch-clamp recordings, the spontaneous action potential firing rate did not differ between cultured and acutely isolated cells, despite modest changes in a subset of action potential waveform parameters. The biophysical properties of two membrane currents that are critical for pacemaker activity in SAMs, the "funny current" (If) and voltage-gated Ca(2+) currents (ICa), were also indistinguishable between cultured and acutely isolated cells. This new method for culture and adenoviral infection of fully-differentiated SAMs from the adult mouse heart expands the range of experimental techniques that can be applied to study the molecular physiology of cardiac pacemaking because it will enable studies in which protein expression levels can be modified or genetically encoded reporter molecules expressed within SAMs.

Culture and adenoviral infection of sinoatrial node myocytes from adult mice.

Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. However, current approaches are inefficient. Here we demonstrate that pro-fibrotic signalling potently antagonizes cardiac reprogramming. Remarkably, inhibition of pro-fibrotic signalling using small molecules that target the transforming growth factor-β or Rho-associated kinase pathways converts embryonic fibroblasts into functional cardiomyocyte-like cells, with the efficiency up to 60%. Conversely, overactivation of these pro-fibrotic signalling networks attenuates cardiac reprogramming. Furthermore, inhibition of pro-fibrotic signalling dramatically enhances the kinetics of cardiac reprogramming, with spontaneously contracting cardiomyocytes emerging in less than 2 weeks, as opposed to 4 weeks with GHMT alone. These findings provide new insights into the molecular mechanisms underlying cardiac conversion of fibroblasts and would enhance efforts to generate cardiomyocytes for clinical applications.

Article	Year
Identification of a region of RyR1 that participates in allosteric coupling with the alpha(1S) (Ca(V)1.1) II-III loop. The Journal of biological chemistry\| PubMed ID: 11726651	2002
Pacemaker channels produce an instantaneous current. The Journal of biological chemistry\| PubMed ID: 11741901	2002
Different roles for the cyclic nucleotide binding domain and amino terminus in assembly and expression of hyperpolarization-activated, cyclic nucleotide-gated channels. The Journal of biological chemistry\| PubMed ID: 12034718	2002
Separable gating mechanisms in a Mammalian pacemaker channel. The Journal of biological chemistry\| PubMed ID: 12121985	2002
A conserved domain in the NH2 terminus important for assembly and functional expression of pacemaker channels. The Journal of biological chemistry\| PubMed ID: 12193608	2002
Inactivation in HCN channels results from reclosure of the activation gate: desensitization to voltage. Neuron\| PubMed ID: 15003173	2004
Distinct populations of HCN pacemaker channels produce voltage-dependent and voltage-independent currents. The Journal of general physiology\| PubMed ID: 16446506	2006
Phosphorylation and modulation of hyperpolarization-activated HCN4 channels by protein kinase A in the mouse sinoatrial node. The Journal of general physiology\| PubMed ID: 20713547	2010
Myristoylated peptides potentiate the funny current (I(f)) in sinoatrial myocytes. Channels (Austin, Tex.)\| PubMed ID: 21150293	2011
Cellular context and multiple channel domains determine cAMP sensitivity of HCN4 channels: ligand-independent relief of autoinhibition in HCN4. The Journal of general physiology\| PubMed ID: 23109717	2012
PKA-independent activation of I(f) by cAMP in mouse sinoatrial myocytes. Channels (Austin, Tex.)\| PubMed ID: 23756695	2013
Complete atrial-specific knockout of sodium-calcium exchange eliminates sinoatrial node pacemaker activity. PloS one\| PubMed ID: 24278453	2013
Culture and adenoviral infection of sinoatrial node myocytes from adult mice. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 26001410	2015
High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling. Nature communications\| PubMed ID: 26354680	2015

Catherine Proenza

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