Kirsten Bender

Sphingosylphosphocholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors.

Blood plasma and serum contain factors that activate inwardly rectifying GIRK1/GIRK4 K+ channels in atrial myocytes via one or more non-atropine-sensitive receptors coupled to pertussis-toxin-sensitive G-proteins. This channel is also the target of muscarinic M(2) receptors activated by the physiological release of acetylcholine from parasympathetic nerve endings. By using a combination of HPLC and TLC techniques with matrix-assisted laser desorption ionization-time-of-flight MS, we purified and identified sphingosine 1-phosphate (SPP) and sphingosylphosphocholine (SPC) as the plasma and serum factors responsible for activating the inwardly rectifying K+ channel (I(K)). With the use of MS the concentration of SPC was estimated at 50 nM in plasma and 130 nM in serum; those concentrations exceeded the 1.5 nM EC(50) measured in guinea-pig atrial myocytes. With the use of reverse-transcriptase-mediated PCR and/or Western blot analysis, we detected Edg1, Edg3, Edg5 and Edg8 as well as OGR1 sphingolipid receptor transcripts and/or proteins. In perfused guinea-pig hearts, SPC exerted a negative chronotropic effect with a threshold concentration of 1 microM. SPC was completely removed after perfusion through the coronary circulation at a concentration of 10 microM. On the basis of their constitutive presence in plasma, the expression of specific receptors, and a mechanism of ligand inactivation, we propose that SPP and SPC might have a physiologically relevant role in the regulation of the heart.

Pannexin 1 Constitutes the Large Conductance Cation Channel of Cardiac Myocytes

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca2+ release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca2+ release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.

A role for RGS10 in β-adrenergic modulation of G-protein-activated K+ (GIRK) channel current in rat atrial myocytes

The effect of β‐adrenergic stimulation on endogenous G‐protein‐activated K+ (GIRK) current has been investigated in atrial myocytes from hearts of adult rats. β‐Adrenergic stimulation (10 μm isoprenaline, Iso) had no effect on activation kinetics, peak current or steady‐state current but resulted in slowing of deactivation upon washout of acetylcholine (ACh), the time constant (τd) being increased by a factor of about 2.5. The effect of Iso could be mimicked by inclusion of cAMP (500 μm) in the filling solution of the patch clamp pipette. The Iso‐induced increase in τd was blocked by the selective β1 receptor antagonist CGP‐20112A (2 μm) and by the PKA inhibitor H9 (100 μm included in the pipette solution). A candidate for mediating these effects is RGS10, one of the regulators of G‐protein signalling (RGS) species expressed in cardiac myocytes. Overexpression of RGS10 by adenoviral gene transfer resulted in a reduction in τd of 60%. Sensitivity of τd to Iso remained in cells overexpressing RGS10. Overexpression of RGS4 caused a comparable reduction in τd, which became insensitive to Iso. Expression of an RGS10 carrying a mutation (RGS10‐S168A), which deletes a PKA phosphorylation site, caused a decrease in τd comparable to overexpression of wild‐type RGS10. Sensitivity of τd to Iso was lost in RGS10‐S168A‐expressing myocytes. Silencing of RGS10 by means of adenovirus‐mediated transcription of a short hairpin RNA did not affect basal τd but removed sensitivity to Iso. These data suggest that endogenous RGS10 has GTPase‐activating protein (GAP) activity on the G‐protein species that mediates activation of atrial GIRK channels. Moreover, RGS10, via PKA‐dependent phosphorylation, enables a crosstalk between β‐adrenergic and muscarinic cholinergic signalling.

Transfection of a phosphatidyl‐4‐phosphate 5‐kinase gene into rat atrial myocytes removes inhibition of GIRK current by endothelin and α‐adrenergic agonists

GIRK (G protein‐activated inward‐rectifying K+ channel) channels, important regulators of membrane excitability in the heart and in the central nervous, are activated by interaction with βγ subunits from heterotrimeric G proteins upon receptor stimulation. For activation interaction of the channel with phosphatidylinositol 4,5‐bisphosphate (PtIns(4,5)P2) is conditional. Previous studies have provided evidence that in myocytes PtIns(4,5)P2 levels relevant to GIRK channel regulation are under regulatory control of receptors activating phospholipase C. In the present study a phosphatidyl‐4‐phosphate 5‐kinase was expressed in atrial myocytes by transient transfection. This did not affect basal properties of GIRK current activated by acetylcholine via M2 receptors but completely abolished inhibition of guanosine triphosphate‐γ‐S activated current by endothelin‐1 or α‐adrenergic agonists. We conclude that though PtIns(4,5)P2 is conditional for channel gating, its normal level in the membrane is not limiting basal function of GIRK channels. Moreover, our data provide further evidence for a regulation of GIRK channels by α1A receptors and endothelin‐A receptors, endogenously expressed in atrial myocytes, via depletion of PtIns(4,5)P2.

G Protein-Activated (GIRK) Current in Rat Ventricular Myocytes is Masked by Constitutive Inward Rectifier Current (I K1 )

Inwardly-rectifying K⁺ channel subunits are not homogenously expressed in different cardiac tissues. In ventricular myocytes (VM) the background current-voltage relation is dominated by I_K1, carried by channels composed of Kir2.x subunits, which is less important in atrial myocytes (AM). On the other hand in AM a large G protein gated current carried by Kir3.1/3.4 complexes can be activated by stimulation of muscarinic M₂ receptors (I_K(ACh)), which is assumed to be marginal in VM. Recent evidence suggests that total current carried by cardiac inward-rectifiers (I_K(ATP), I_K(ACh), I_K1) in both, AM and VM is limited, due to K⁺ accumulation/depletion. This lead us to hypothesize that in conventional whole celI recordings I_K(ACh) in VM is underestimated as a consequence of constitutive I_K1. In that case a reduction in density of I_K1 should be paralleled by an increase in density of I_K(ACh). Three different experimental strategies have been used to test for this hypothesis: (i) Adenovirus-driven expression of a dominant-negative mutant of Kir2.1, one of the subunits supposed to form I_K1 channels, in VM caused a reduction in I_K1-density by about 80 %. In those cells I_K(ACh) was increased about 4 fold. (ii) A comparable increase in I_K(ACh) was observed upon reduction of I_K1 caused by adenovirus-mediated RNA interference.(iii) Ba²⁺ in a concentration of 2 µM blocks I_K1 in VM by about 60 % without affecting atrial I_K(ACh). The reduction in I_K1 by 2 µM Ba²⁺ is paralleled by a reversible increase in I_K(ACh) by about 100%. These data demonstrate that the increase in K⁺ conductance underlying ventricular I_K(ACh) is largely underestimated, suggesting that it might be of greater physiological relevance than previously thought.

Generation of a constitutive Na+‐dependent inward‐rectifier current in rat adult atrial myocytes by overexpression of Kir3.4

Apart from gating by interaction with βγ subunits from heterotrimeric G proteins upon stimulation of appropriate receptors, Kir.3 channels have been shown to be gated by intracellular Na+. However, no information is available on how Na+‐dependent gating affects endogenous Kir3.1/Kir3.4 channels in mammalian atrial myocytes. We therefore studied how loading of adult atrial myocytes from rat hearts via the patch pipette filling solution with different concentrations of Na+ ([Na+]pip) affects Kir3 current. Surprisingly, in a range between 0 and 60 mm, Na+ neither had an effect on basal inward‐rectifier current nor on the current activated by acetylcholine. Overexpression of Kir3.4 in adult atrial myocytes forced by adenoviral gene transfer results in formation of functional homomeric channels that interact with βγ subunits upon activation of endogenous muscarinic receptors. These channels are activated at [Na+]pip≥ 15 mm, resulting in a receptor‐independent basal inward rectifier current (Ibir). Ibir was neither affected by pertussis toxin nor by GDP‐β‐S, suggesting G‐protein‐independent activation. PIP2 depletion via endogenous PLC‐coupled α1 adrenergic receptors causes inhibition of endogenous Kir3.1/3.4 channel currents by about 75%. In contrast, inhibition of Na+‐activated Ibir amounts to < 20%. The effect of the Kir3 channel blocker tertiapin‐Q can be described using an IC50 of 12 nm (endogenous IK(ACh)) and 0.61 nm (Ibir). These data clearly identify Ibir as a homotetrameric Kir3.4 channel current with novel properties of regulation and pharmacology. Ibir shares some properties with a basal current recently described in atrial myocytes from an animal model of atrial fibrillation (AF) and AF patients.

Coupling to Gs and Gq/11 of histamine H2 receptors heterologously expressed in adult rat atrial myocytes

The predominant histamine receptor subtype in the supraventricular and ventricular tissue of various mammalian species is the H2 receptor (H2-R) subtype, which is known to couple to stimulatory G proteins (Gs), i.e. the major effects of this autacoid are an increase in sinus rate and in force of contraction. To investigate histamine effects in H2-R-transfected rat atrial myocytes, endogenous GIRK currents and L-type Ca2+ currents were used as functional assays. In H2-R-transfected myocytes, exposure to His resulted in a reversible augmentation of L-type Ca2+ currents, consistent with the established coupling of this receptor to the Gs-cAMP-PKA signalling pathway. Mammalian K+ channels composed of GIRK (Kir3.x) subunits are directly controlled by interaction with betagamma subunits released from G proteins, which couple to seven-helix receptors. In mock-transfected atrial cardiomyocytes, activation of muscarinic K+ channels (IK(ACh)) was limited to Gi-coupled receptors (M2R, A1R). In H2-R-overexpressing cells, histamine activated IK(ACh) via Gs-derived betagamma subunits since the histamine-induced current was insensitive to pertussis toxin. These data indicate that overexpression of Gs-coupled H2-R results in a loss of target specificity due to an increased agonist-induced release of Gs-derived betagamma subunits. When IK(ACh) was maximally activated by GTP-gamma-S, histamine induced an irreversible inhibition of the inward current in a fraction of H2-R-transfected cells. This inhibition is supposed to be mediated via a G(q/11)-PLC-mediated depletion of PIP2, suggesting a partial coupling of overexpressed H2-R to G(q/11). Dual coupling of H2-Rs to Gs and Gq is demonstrated for the first time in cardiac myocytes. It represents a novel mechanism to augment positive inotropic effects by activating two different signalling pathways via one type of histamine receptor. Activation of the Gs-cAMP-PKA pathway promotes Ca2+ influx through phosphorylation of L-type Ca2+ channels. Simultaneous activation of Gq-signalling pathways might result in phosphoinositide turnover and Ca2+ release from intracellular stores, thereby augmenting H2-induced increases in [Ca2+]i.

Antisense oligonucleotides against receptor kinase GRK2 disrupt target selectivity of β‐adrenergic receptors in atrial myocytes

K+ channels composed of GIRK subunits are predominantly expressed in the heart and various regions of the brain. They are activated by βγ‐subunits released from pertussis toxin‐sensitive G‐proteins coupled to different seven‐helix receptors. In rat atrial myocytes, activation of K(ACh) channels is strictly limited to receptors coupled to pertussis toxin‐sensitive G‐proteins. Upon treatment of myocytes with antisense oligodesoxynucleotides against GRK2, a receptor kinase with Gβγ binding sites, in a fraction of cells, K(ACh) channels can be activated by β‐adrenergic receptors. Sensitivity to β‐agonist is insensitive to pertussis toxin treatment. These findings demonstrate a potential role of Gβγ binding proteins for target selectivity of G‐protein‐coupled receptors

Acute desensitization of GIRK current in rat atrial myocytes is related to K+ current flow

We have investigated the acute desensitization of acetylcholine‐activated GIRK current (IK(ACh)) in cultured adult rat atrial myocytes. Acute desensitization of IK(ACh) is observed as a partial relaxation of current with a half‐time of < 5 s when muscarinic M2 receptors are stimulated by a high concentration (> 2 μmol l−1) of ACh. Under this condition experimental manoeuvres that cause a decrease in the amplitude of IK(ACh), such as partial block of M2 receptors by atropine, intracellular loading with GDP‐β‐S, or exposure to Ba2+, caused a reduction in desensitization. Acute desensitization was also identified as a decrease in current amplitude and a blunting of the response to saturating [ACh] (20 μmol l−1) when the current had been partially activated by a low concentration of ACh or by stimulation of adenosine A1 receptors. A reduction in current analogous to acute desensitization was observed when ATP‐dependent K+ current (IK(ATP)) was activated either by mitochondrial uncoupling using 2,4‐dinitrophenole (DNP) or by the channel opener rilmakalim. Adenovirus‐driven overexpression of Kir2.1, a subunit of constitutively active inwardly rectifying K+ channels, resulted in a large Ba2+‐sensitive background K+ current and a dramatic reduction of ACh‐activated current. Adenovirus‐driven overexpression of GIRK4 (Kir3.4) subunits resulted in an increased agonist‐independent GIRK current paralleled by a reduction in IK(ACh) and removal of the desensitizing component. These data indicate that acute desensitization depends on K+ current flow, independent of the K+ channel species, suggesting that it reflects a reduction in electrochemical driving force rather than a bona fide signalling mechanism. This is supported by the observation that desensitization is paralleled by a significant negative shift in reversal potential of IK(ACh). Since the ACh‐induced hyperpolarization shows comparable desensitization properties as IK(ACh), this novel current‐dependent desensitization is a physiologically relevant process, shaping the time course of parasympathetic bradycardia.

A genetically encoded tool kit for manipulating and monitoring membrane phosphatidylinositol 4,5-bisphosphate in intact cells.

Background Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P2 in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane. For controlled and reversible depletion of PtdIns(4,5)P2, voltage-sensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-Cδ1 (PLCδ1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes. Methods and Results Expression and correct targeting of ECFP-PH-PLCδ1, EYFP-PH-PLCδ1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of PtdIns(4,5)P2 in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated K+ (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdIns(4,5)P2 was identified. Conclusions Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.