SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

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.

G protein-activated inwardly rectifying K+ channels, comprised of Kir3.x subunits are expressed in the heart, in central and peripheral neurons, various endocrine tissues, but also in non-excitable structures such as blood platelets (Shankar et al. 2004). They contribute to regulating cardiac frequency, hormone secretion and mediate slow inhibitory postsynaptic potentials (Kaupmann et al. 1998; Inanobe et al. 1999; Stanfield et al. 2002; Koyrakh et al. 2005). These channels are activated by direct interaction with βγ subunits derived from pertussis toxin-sensitive heterotrimeric G proteins upon stimulation of appropriate G protein-coupled receptors (GPCR) (Dascal, 2001; Sadja et al. 2003). In the heart, functional Kir3 channels are expressed preferentially but not exclusively in the sinoatrial node, atrioventricular node and in atrial myocytes (Dobrzynski et al. 2002; Marionneau et al. 2005). Apart from the paradigmatic muscarinic M2 receptor, other endogenous GPCR species that couple to pertussis toxin-sensitive G proteins can activate cardiac Kir3 channels such as purinergic A1 receptors (Kurachi et al. 1986; Bünemann & Pott, 1995) and sphingolipid receptors (Bünemann et al. 1996).

Besides the canonical regulation by Gβγ, evidence for modulation of Kir3 channels by other factors has been demonstrated, such as the regulator of G protein signalling (RGS) proteins (Doupnik et al. 1997; Ishii et al. 2002; Fu et al. 2006), the anionic phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) (Cho et al. 2001, 2005; Sadja et al. 2003), and intracellular Mg2+ and Na+ concentrations (Petit-Jacques et al. 1999). Two distinct mechanisms have been described, by which [Na+]i contributes to regulation of Kir3 channels. Strong evidence has been provided for direct activation by interaction with Na+-binding aspartate residues in the C-terminus of cardiac Kir3.4 or neuronal Kir3.2 subunits (Lesage et al. 1995; Sui et al. 1996; Ho & Murrell-Lagnado, 1999). In addition, it has been described that Na+ in a physiological range of concentrations promotes dissociation or re-arrangement, respectively (Bünemann et al. 2003) of GαGDP and Gβγ, which in turn affects gating kinetics of Kir3 channels (Rishal et al. 2003; Yakubovich et al. 2005). Direct activation of Kir3 channels by [Na+]i is supposed to be independent of Gβγ binding, but to require PIP2 (Ho & Murrell-Lagnado, 1999). In cardiac tissue, frequency-dependent changes in global or subsarcolemmal [Na+]i are likely to occur. Therefore [Na+]i-dependent modulation of Kir3 current might represent a physiological feedback mechanism, that could contribute to controlling cardiac frequency and excitability. In the present study, the effects of [Na+] in the pipette filling solution ([Na+]pip) on whole-cell current carried either by endogenous Kir3.1/Kir3.4 channels or by homomeric Kir3.4 channels after adenovirus-driven overexpression of Kir3.4 have been investigated in cultured adult atrial myocytes in a range of concentrations between nominally zero and 60 mm.

[Na+]pip did not affect basal (agonist-independent) activation of endogenous Kir3 channels. In myocytes transformed with Ad-Kir3.4, at [Na+]pip≥ 15 mm, a K+ current activated spontaneously upon getting into the whole-cell mode. From its current–voltage characteristics, the basal current (Ibir) could be identified as being carried by homomeric Kir3.4 channels (Bender et al. 2001). Since endogenous current in atrial myocytes is supposed to be carried exclusively by Kir3.1/Kir3.4 tetrameric complexes (Kennedy et al. 1999), direct [Na+]i-dependent regulation of cardiac Kir3 channels obviously has little relevance under normal physiological conditions, but might be relevant in the process of electrical remodelling associated with chronic atrial fibrillation (e.g. Dobrev et al. 2005; Cha et al. 2006).

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Isolation and culture of atrial myocytes

Rats were killed following protocols approved by the local authorities for the regulation of animal welfare (Regierungspräsident) in accordance with the guidelines of the European Community (86/609/EEC). Wistar Kyoto rats of either sex, aged between 8 and 10 weeks, were anaesthetized by i.v. injection of urethane (1 g kg−1). Following thoracotomy, the heart was quickly removed and mounted on a modified Langendorff apparatus for perfusion at constant flow under sterile conditions. The method of enzymatic isolation of atrial myocytes from hearts of adult rats and serum-free culture conditions have been described elsewhere (e.g. Bünemann et al. 1997). As a rule, myocytes were used experimentally for 5–7 days after isolation.

Solutions and chemicals

For whole-cell measurements of membrane currents, an extracellular solution of the following composition was used (mm): NaCl 120; KCl 20; CaCl2 0.5; MgCl2 1.0; Hepes/NaOH 10.0, pH 7.4. The standard pipette solution contained (mm): potassium aspartate 100; KCl 40.0; NaCl 5.0, MgCl2 7.0; Na2ATP 5.0; EGTA 2.0; GTP 0.025; Hepes/KOH 20.0, pH 7.4. The K+ reversal potential under these conditions was calculated as −50 mV. Total Na+ concentration of this solution was 15 mm. Pipette solutions containing different Na+ concentrations (0–60 mm) were made up by corresponding changes in [NaCl] compensated for by equimolar changes in [KCl] and replacement of Na2ATP by the K+ salt. Standard chemicals were from Merck (Darmstadt, Germany). EGTA, Hepes, Na2ATP, K2ATP, GTP, GTP-γ-S, GDP-β-S, acetylcholine chloride (ACh) and pertussis toxin (PTX) were from Sigma (Deisenhofen, Germany). Tertiapin-Q (T-Q) was from Tocris (Ellisville MI, USA).

Whole-cell patch clamp recording

Membrane currents were measured at ambient temperature (22–24°C) using the whole-cell patch clamp technique (Bünemann et al. 1997). Cells were routinely clamped at a holding potential of −90 mV, i.e. negative to EK, resulting in inward K+ currents. Current–voltage relations were determined by means of voltage ramps between −120 mV and +60 mV within 500 ms, applied at 0.1 s−1. Rapid exposure to solutions containing ACh and other compounds was performed by means of a custom-made solenoid-operated flow system.

Adenovirus constructs and gene transfer in atrial myocytes

The pAd-Easy1 plasmid encoding for the adenovirus type 5 and pAd-Track-CMV were kindly provided by Dr B. Vogelstein (Johns Hopkins University, Baltimore, MD, USA). Production and purification of the recombinant virus were performed as described in detail previously (He et al. 1998). Briefly, the cDNA of rat Kir3.4 (obtained from Dr Y. Kurachi, Osaka, Japan) was subcloned into pAd-Track-CMV shuttle vector, using XbaI and KpnI to yield pAdTrack-Kir3.4. Adenovirus recombinant plasmid was generated by homologous recombination between pAdTrack-Kir3.4 and pAd-Easy-1 in E. coli to produce the recombinant virus. The recombinant viruses were propagated in HEK293 cells and recovered after several freezing–thawing cycles. Virus titres were estimated by serial dilution and infection of myocyte cultures.

Kir3.4 C-terminally fused to yellow fluorescent protein (YFP) was generated by means of cloning PCR-amplificated cDNA into the pEYFP-N1 vector (Clontech). Gβ1-CFP in pcDNA3 was kindly provided by Dr M. Bünemann, Würzburg, Germany. Adenoviral constructs were generated as described above using the pShuttle-CMV as shuttle vector.

For infection, cells were incubated for 3 h, starting 24 h after plating, with 1 ml culture medium containing approximately 106 infectious particles. Electrophysiological recordings were made on days 4–6 after exposure to the virus. Infected cells (as a rule > 50%) were identified by epifluorescence of green fluorescent protein (GFP) or YFP in case of fluorescence resonance energy transfer (FRET) measurements. Time-matched GFP-positive cells infected with the empty GFP-encoding virus served as controls.

FRET measurements

Myocytes or HEK293 cells were plated on polyethylenimine-treated glass coverslips. FRET measurements were performed using an Axiovert 200 inverted microscope (Zeiss, Jena, Germany) equipped with an oil immersion ×100 objective (Planapochromat NA 1.4), a dual-emission photometric system and a polychrome V (both TILL Photonics, Planegg, Germany). To minimize photobleaching, illumination time was ≤ 10 ms at a frequency of 3 Hz. Excitation wavelength was set to 435 ± 10 nm (beam splitter DCLP 460 nm), and emission was recorded at 515 nm (emission filter LP 515 nm) and 480 ± 20 nm (beam splitter DCLP 505 nm). YFP emission was corrected for direct excitation (YFP emission at 435 nm excitation/YFP emission at 505 nm excitation was 0.17) and spillover of CFP into the 535 nm channel.

Statistical analysis

Summarized data are presented as mean ±s.e.m. and differences were analysed using Student's unpaired t test. P < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Membrane localization of Kir3.4 and agonist–induced interaction with Gβ2

In the literature there is overwhelming evidence that cardiac Kir3 channels are composed in a heterotetrameric fashion of Kir3.1 and Kir3.4 (see Stanfield et al. 2002 for review). Evidence for functional Kir3.4 homomeric channels in myocytes has been provided, when these were overexpressed. Moreover, in CHO cells expression of Kir3.4 without Kir3.1 resulted in robust currents that can be activated by a co-expressed GPCR (Bender et al. 2001). On the other hand, in various studies using Xenopus oocytes expressing wild-type Kir3.4, little or no inward-rectifying current could be observed, suggesting poor membrane translocation and functionality. In order to demonstrate membrane translocation and GPCR-induced interaction of Kir3.4 with Gβγ, we co-infected atrial myocytes with adenoviral vectors containing cDNA for the Kir3.4–YFP and β2–CFP fusion constructs. A representative result from these experiments is illustrated in Fig. 1. In line with previous studies, adult atrial myocytes, in contrast to ventricular cells under identical conditions, attain a spherical shape within a few hours of isolation (Fig. 1A; cf. Mechmann & Pott, 1986) but retain their major properties for up to about 2 weeks in vitro. A clear membrane localization of the tagged Kir3.4 can be identified (Fig. 1C).

image

Figure 1. FRET measurements demonstrating membrane localization of Kir3.4 fused to YFP and agonist-induced interaction with CFP-tagged Gβ2 A, phase-contrast photomicrograph of cultured atrial myocytes (4 days in culture). The cell marked by the asterisk is a contaminating ventricular myocyte. B, schematic representation of the experimental design. The Gβγ2–CFP fusion construct is assumed to associate with endogenous Gγ; the prenyl membrane anchor of the γ subunit targets the complex to the membrane and associates with Gα (not shown for clarity). Upon stimulation of an appropriate GPCR the βγ complex binds to Kir3.4. C, representative fluorescence images of an atrial myocyte demonstrating – apart from intracellular localization of the fluorescent fusion proteins in unidentified compartments – their targeting to the membrane. D, changes in fluorescence of CFP and YFP and resulting FRET ratio induced by application of adenosine in a HEK293 cell transfected with plasmids encoding for Kir3.4–CFP, β2–YFP and a rat A1 adenosine receptor. E, changes in fluorescence evoked by application of ACh (10 μm) to an atrial myocyte infected with adenoviruses encoding for Kir3.4–YFP and β2–CFP. The FRET changes represented in D and E are representative for four independent measurements.

Download figure to PowerPoint

When the fluorescent fusion proteins were co-expressed with an adenosine A1 receptor in HEK293 cells, which are devoid of endogenous Kir3 subunits, robust FRET signals could be activated by adenosine (Fig. 1D). Correspondingly, Ado-induced Kir3.4 currents could be recorded from these cells (not shown).

In atrial myocytes expressing the fluorescent fusion constructs a rapid increase in YFP fluorescence paralleled by a decrease in CFP fluorescence was observed upon stimulation of the endogenous M2 receptors by 10 μm acetylcholine (Fig. 1E). The resulting positive shift in FRET ratio clearly indicates an interaction of Kir3.4 with Gβγ. Although the signals in myocytes might be contaminated by FRET from Kir3.1–Kir3.4 heteromers interacting with Gβγ, these results confirm that Kir3.4 forms functional homomeric channels that can be activated by Gβγ.

Effects of [Na+]pip on endogenous Kir3.1–Kir3.4 current and current carried by Kir3.4 homotetramers

The current traces in Fig. 2A were recorded from myocytes infected with the empty virus at different Na+ concentrations in the pipette filling solution, ranging from nominally 0 mm to 60 mm. These recordings include the transition from the cell-attached mode to the whole-cell recording configuration. The representative traces neither reveal any obvious effect of the different pipette solutions on IK(ACh) activated by rapid exposure to a saturating concentration of ACh (10 μm) nor Ba2+-sensitive (1 mm) basal current. The degree of acute desensitization is also in the normal range of variability. Comparable results were also obtained from native myocytes cultured for a few hours and up to 6 days (not shown). Figure 2B shows analogous recordings from myocytes infected with Ad-Kir3.4. In line with published data, activation of IK(ACh) in these cells is slower (Bender et al. 2001; Sadja et al. 2002) and lacks fast desensitization (Bender et al. 2004). The more salient difference to mock-infected or native cells, however, is the development of an inward current within about 1 min after rupturing the patch at [Na+]pip= 15 mm and which is more pronounced at 60 mm. In the following, this current will be termed Ibir (basal inward rectifying, see Fig. 3) as a distinction from the current activated by ACh (IK(ACh)).

image

Figure 2. Effects of different [Na+]pip on Ba2+-sensitive basal current and IK(ACh) A, representative current recordings from myocytes infected with empty virus (for experimental details see Methods). In this and subsequent figures, the arrows indicate the transition from the cell-attached to the whole-cell mode. Solutions containing ACh (10 μm) or BaCl2 (1 mm) were applied as indicated. The current that remained in the presence of 1 mm Ba2+ was defined as basal current. B, analogous current traces recorded from myocytes transformed with Ad-Kir3.4 5 days after exposure to the virus. The rapid vertical deflections in this and subsequent figures represent changes in membrane current caused by voltage ramps from −120 to +60 mV that were routinely applied at 0.1 s−1. C, normalized fractions of basal current (Ibir) and ACh-activated current to total current as function of [Na+]pip. Each symbol is based on 10 measurements.

Download figure to PowerPoint

image

Figure 3. Comparison of inward rectification of Ibir and IK(ACh) in Kir3.4-overexpressing myocytes versus native IK(ACh) A, representative recording of basal current and IK(ACh) from a myocyte infected with the empty virus. B, analogous recording from an Ad-Kir3.4-infected cell. Pipette filling solution contained 40 mm Na+ in both experiments. C, current–voltage curves from A and B (leak current-subtracted as indicated by the lowercase letters; no correction for liquid junction potential was applied). D, ratios of current at +50 mV and at −100 mV have been summarized to quantify inward rectification (n= 5).

Download figure to PowerPoint

The concentration–response curve for [Na+]pip (Fig. 2C) reveals a non-saturating increase in Ibir over the range of concentrations tested, paralleled by a decrease in IK(ACh). The reduction in the amplitude of receptor-activated IK(ACh) possibly reflects (i) a gradual loss in channels available to canonical activation by Gβγ due to receptor-independent gating and (ii) limitation of total K+ current flow due to accumulation/depletion phenomena (Bender et al. 2004; Wellner-Kienitz et al. 2004). No activation of Ibir was observed, when Na+ (60 mm) was replaced by Li+ (not shown), demonstrating that this current is a consequence of raising [Na+]pip, rather than reducing [K+]pip

Voltage dependence of Ibir and IK(ACh)

We next compared the inward rectifying properties of Kir3 currents in mock-infected and Ad-Kir3.4-infected myocytes. Two representative traces recorded with 40 mm[Na+]pip are shown in Fig. 3A (mock infected) and B (Ad-Kir3.4-infected). The current–voltage relation of native IK(ACh) was obtained by subtraction of ramp currents labelled a and b in A and plotted in Fig. 3C. It is characterized by strong inward rectification. The current–voltage relations of Ibir and IK(ACh) from the Ad-Kir3.4 transduced cell were extracted analogously and also plotted in Fig. 3C. Both Ibir and IK(ACh) in the Ad-Kir3.4-transformed cells show less inward rectification than native IK(ACh). The summarized data in Fig. 3D represent ratios of current levels at +50 mV and −100 mV as a measure of inward rectification. These ratios for Ibir and IK(ACh) from the Kir3.4-infected myocytes were significantly larger than for native IK(ACh), i.e. the currents in Kir3.4-overexpressing cells showed less inward rectification as compared with endogenous IK(ACh). Thus, the rectifying property of Ibir is similar to that of ACh-activated Kir3.4 homotetrameric channels previously described (Bender et al. 2001)

Activation of Ibir by Na+ is independent of the G protein cycle

Activation of expressed Kir3 channels by [Na+]i in oocytes has been shown to be independent of Gβγ (Petit-Jacques et al. 1999; Ho & Murrell-Lagnado, 1999). To investigate, if this applies to activation of Ibir, Ad-Kir3.4-transformed myocytes were incubated with pertussis toxin (PTX) using a protocol that has been shown to result in complete suppression of receptor-activated Kir3 current in atrial myocytes (Wellner-Kienitz et al. 2001). Figure 4A shows a representative current trace recorded from a PTX-treated Ad-Kir3.4-infected myocyte using a pipette solution containing 40 mm Na+. Analogous to untreated cells, a robust inward current was activated upon rupturing the membrane patch. A challenge by 10 μm ACh on the plateau of Ibir failed to elicit additional current, demonstrating complete functional knockdown of the G protein species that couples to Kir3 channels. This was highly representative as demonstrated by the summarized data (Fig. 4B). Stimulation of muscarinic receptors even resulted in a slight but significant reduction in Ibir in PTX-treated cells. Independence of Ibir on intact G protein cycling was corroborated in a series of experiments using a pipette solution in which GTP was replaced by GDP-β-S, an analogue that is poorly phosphorylated causing accumulation of heterotrimeric protein complexes not susceptible to activation by GPCR stimulation. As illustrated in Fig. 4C and D, robust activation of Ibir was recorded, which remained stable over more than 4 min, whereas IK(ACh) was attenuated by more than 50% within this period of time.

image

Figure 4. Inhibition of G protein cycling does not affect Ibir A, current recording from a myocyte that was incubated with PTX. [Na+]pip was 40 mm in this series of experiments. B, summarized data demonstrating insensitivity of Ibir, defined as Ba2+-sensitive current, to PTX using conditions that caused complete inhibition of IK(ACh) (n= 5). C, recording of membrane current using a pipette filling solution that contained 1 mm GDP-β-S ([Na+]pip= 60 mm). Myocytes were not treated with PTX in this series of experiments. For the first challenge by ACh a time was chosen when Ibir was about to reach a steady state. The amplitude of the resulting IK(ACh) served as reference for subsequent ACh-evoked currents. D, summarized data from this series of experiments reveal a significant reduction in IK(ACh) within about 100 s with regard to the first response. No significant loss in Ibir was observed within that period of time (n= 5).

Download figure to PowerPoint

Sensitivity of Ibir to receptor-mediated depletion of PIP2

Kir3 channels, like a number of other ion channels and transporters, are either regulated by PIP2 or require a certain membrane concentration of this anionic phospholipid in their microenvironment for normal function (Takano & Kuratomi, 2003). It has been suggested that activation by both Gβγ and [Na+]i proceeds via stabilization of PIP2 binding to the channel (Huang et al. 1998; Zhang et al. 1999).

In rat atrial myocytes membrane concentrations of PIP2 can be reduced by stimulation of endogenous receptors activating phospholipase C (PLC), such as the α1-adrenergic receptor or the ETA endothelin receptor. Activation of these receptors has been demonstrated to result in inhibition of IK(ACh) in atrial myocytes (Cho et al. 2001, 2005; Meyer et al. 2001; Bender et al. 2002). With standard GTP-containing pipette solution, stimulation of α1-receptors induces a transient activation of IK(ACh), presumably due to partial coupling of these receptors to Gi/o (Meyer et al. 2001), which is superimposed on the inhibition reflecting PIP2 depletion. Therefore, following previous studies using comparable conditions, we used a GTP-γ-S-loading protocol to assess the effects of receptor-induced PIP2 depletion on Ibir. Figure 5A shows a representative current recording from a native cell to demonstrate the effect of GTP-γ-S loading, which, accelerated by brief exposures to ACh, resulted in a stable activation of IK(ACh). Phenylephrine (100 μm) caused a reduction of this current by about 70% on average (Fig. 5E), in line with published data (Meyer et al. 2001; Bender et al. 2002). A significantly stronger inhibition of IK(ACh) (∼85%) was obtained in myocytes overexpressing Kir3.4 at 0 mm[Na+]pip (Fig. 5B and E). In contrast, Ibir activated by 60 mm[Na+]pip, without GTP-γ-S in the pipette solution, showed little sensitivity to α1-receptor-mediated depletion of PIP2 (Fig. 5C and E). Although small (24%), this reduction in Ibir could be at least partially attributed to stimulation of α1-receptors, since spontaneous rundown was < 10% (Fig. 5D and E). Thus, Kir3.4 homomeric channels activated by Na+ can be distinguished from those activated by Gβγ by a much smaller inhibition by PLC-induced PIP2 depletion. This does not imply that Kir3.4 channels do not require PIP2 for Na+-dependent gating. More probably, the low sensitivity of Ibir reflects a higher affinity of PIP2 to the Na+-gated mode of the channel as compared with the Gβγ-bound channel complex.

image

Figure 5. Differential sensitivities of IK(ACh) and Ibir to phenylephrine-induced depletion of PIP2 A–D, representative recordings of membrane current: A, control (empty virus-infected) myocyte loaded with GTP-γ-S (500 μm) via the patch pipette. ACh and phenylephrine were applied as indicated. B, C and D, Ad-Kir3.4-infected cells. The trace in B (Na+-free pipette solution, supplemented with GTP-γ-S) represents the Kir3.4 current activated by Gβγ, whereas C (60 mm[Na+]pip– no GTP-γ-S) represents Na+-activated Ibir. D (no treatment with phenylephrine) is to demonstrate that Ibir shows very little rundown. E, summarized data (n= 10 for each protocol).

Download figure to PowerPoint

Differential sensitivities of IK(ACh) and Ibir to tertiapin-Q

The bee venom peptide tertiapin or its more stable synthetic derivative tertiapin-Q, respectively, represent standard compounds to pharmacologically identify a current as being carried by Kir3-related channels (Jin & Lu, 1999; Kitamura et al. 2000). T-Q has been demonstrated to block endogenous and reconstituted Kir3.1–Kir3.4 channels with an EC50 of about 10 nm, whereas other inward rectifiers expressed in cardiac myocytes (IK1– Kir2.1–Kir2.x, and IK(ATP)– Kir6.2–SUR2A) are more than 100-fold less sensitive (Jin & Lu, 1998; Kitamura et al. 2000). In order to further characterize the nature of Ibir, sensitivities to T-Q of IKACh) in native cells and IK(ACh) and Ibir in Ad-Kir3.4-transformed cells were compared. Representative current traces depicting the inhibitory actions of T-Q on Kir3 currents are shown in Fig. 6A and B and summarized as concentration–response curves in Fig. 6C. Sensitivity of the native current (EC50= 12 nm, Fig. 6C) is comparable to published data. A significantly higher affinity of T-Q was found for the receptor-activated current in Ad-Kir3.4-transformed cells (EC50= 1.4 nm, Fig. 6C). The blocking action on Na+-dependent Ibir in Ad-Kir3.4-infected cells was characterized by an EC50 of 0.6 nm. This implies that the affinity of Kir3.4 homomeric channels to T-Q is more than one order of magnitude higher as compared with endogenous Kir3.1–Kir3.4 channels. The difference between EC50 values for receptor-activated and Na+-activated current were not significant, suggesting that the affinity of the blocker is determined by the subunit composition rather than by the mechanism of gating.

image

Figure 6. Block of IK(ACh) and Ibir by T-Q A, representative recording of membrane current from a control (empty virus) transformed cell. ACh and T-Q were applied as indicated. B, representative recording of Ibir activated by 60 mm[Na+]pip in an Ad-Kir3.4-transformed cell. C, concentration–response curves. For comparison the curve reflecting inhibition of IK(ACh) in Ad-Kir3.4 cells has been included. Data were fitted using simple saturation kinetics. Each data point represents the mean value ±s.e.m. of 4–6 measurements.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Apart from canonical activation by direct interaction with Gβγ upon stimulation of a GPCR, Kir3 channels have been shown to be regulated by a variety of other factors such as RGS proteins, PIP2 and [Na+]i (see Stanfield et al. 2002; Sadja et al. 2003 for reviews). Regulation of Kir3 channels by PIP2 has been demonstrated convincingly in expression systems but also in the physiological context of adult atrial myocytes by activation of endogenous PLC-coupled receptors resulting in PIP2 depletion via a physiological pathway (Cho et al. 2001, 2005; Meyer et al. 2001; Bender et al. 2002). In contrast, very little is known about the physiological role of [Na+]i-dependent gating and its relation to activation of Kir3 current by endogenous G protein-coupled receptors in cardiac myocytes, neurons and other differentiated mammalian cells that endogenously express Kir3 channels.

The initial aim of the present study therefore was to characterize the regulation of endogenous Kir3.1–Kir3.4 channels in atrial myocytes by Na+-dependent gating and how this relates to canonical activation by stimulation of muscarinic M2 receptors. The major result of the present study was the lack of an effect of [Na+]pip on basal and receptor-evoked current carried by endogenous Kir3.x channels in rat atrial myocytes at a range of concentrations between 0 mm and 60 mm. Under otherwise identical conditions robust activation of a basal inwardly rectifying current by [Na+]pip was found in myocytes overexpressing Kir3.4.

The existence and functionality of homomeric wild-type Kir3.4 channels is not undisputed. In a number of studies, clearly defined single channel events could not be identified in oocytes injected with Kir3.4 RNA. This led to the conclusion that homomeric complexes of wild-type Kir3.4 are not functional. However, a publication by Ji et al. (1998) reported that defined unitary currents carried by Kir3.4 homomers could be resolved but ran down rapidly in inside-out patches from oocytes. The reason for this instability is unknown. In mammalian cells – atrial myocytes and HEK293 cells – clear evidence for stable macroscopic currents carried by homomeric Kir3.4 channels has been provided (Bender et al. 2001). Kir3.4 homomeric channels could be detected at a low level in Kir3.1 knockout mice (Bettahi et al. 2002). Their contribution to the endogenous GPCR-activated current was considered marginal by these authors.

Current carried by endogenous Kir3.1–Kir3.4 channels and Kir3.4 homomeric channels following overexpression of Kir3.4 or dimeric or tetrameric constructs, respectively, could be discriminated by differences in inward rectification, which was significantly weaker in the latter. Comparable inward-rectifying properties of Kir3.4 channel currents could be reproduced in HEK293 cells transfected with vectors encoding for Kir3.4 or Kir3.4 dimers or tetramers (Bender et al. 2001). These findings combined with the FRET data of the present study leave no doubts that Kir3.4 subunits are translocated to the surface membrane as homomeric channels and functionally interact with βγ subunits upon stimulation of an appropriate GPCR.

Co-transfection of Kir3.1 in the study by Bender et al. (2001) resulted in currents with stronger rectification, comparable to native atrial myocytes. No basal activation of an inwardly rectifying current was reported in (Kir3.4)x-transfected myocytes and HEK293 cells, which most probably reflects the fact that the standard Na+ concentration of the pipette filling solution used in that study was lower (9 mm) as compared with the present study (15 mm).

The different degrees in inward-rectification between currents in native myocytes and Ad-Kir3.4-transformed cells suggests that in the former the current is dominated or even exclusively carried by Kir3.1–Kir3.4 heterotetramers. In bovine atrial muscle, Kir3.4 homotetrameric protein complexes have been detected (Corey & Clapham, 1998). Whether they contribute to Kir3 currents, either basal or GPCR-activated, in that species or other species is not known.

Activation by [Na+]i previously has been found to be independent of Gβγ or the G protein cycle. This also applies to Ibir characterized in the present study, which is insensitive to GDP-β-S and to pertussis toxin.

The low sensitivity of Na+-activated Ibir to PIP2 appears to be contradictory to the findings that activation of Kir3 channels by cytosolic Na+ requires the presence of PIP2 (Sui et al. 1998; Ho & Murrell-Lagnado, 1999). It is assumed that neutralizing the C-terminal aspartate residue by Na+ enables the binding of negatively charged PIP2 to nearby arginine residues. This model implies that Na+-dependent gating is mediated by PIP2. The lower sensitivity to PIP2 of Na+-activated channel current as compared with βγ-activated current, in line with this hypothesis, could be due to a higher affinity of the former to PIP2.

Apart from regulation of Kir3 channel gating by binding of Na+ to Kir3.4 and Kir3.2, an indirect modulation via an accelerating effect of Na+ on the G protein cycle has been demonstrated using reconstitution of the pathway in Xenopus oocytes (Rishal et al. 2003; Yakubovich et al. 2005). This regulation is operating in the physiological range of [Na+]i. It should be independent of the subunit composition of Kir3 channels and therefore should operate also in native myocytes. We assume that different experimental conditions are required to demonstrate and analyse the importance of this mechanism for regulation of endogenous cardiac Kir3 channels by GPCRs. In the present study, a concentration of ACh was used routinely that is highly saturating with regard to current amplitude and kinetics of activation. Putative effects of [Na+]i that accelerate the signal via an effect on the G protein cycle are likely to be blunted under these conditions.

Is Na+-dependent activation of Kir3.4 channels of physiological relevance?

Our results clearly suggest that regulation of endogenous cardiac Kir3 channels in atrial myocytes by [Na+]i has little, if any, relevance under normal physiological conditions. This appears to be contradictory to previous investigations using the Xenopus oocyte expression system or cardiac myocytes from embryonic chick hearts. It is usually assumed that injection of mRNAs for Kir3.1 and Kir3.4 (Kir3.2) into oocytes in a 1 : 1 relation results in a corresponding expression of both subunits and furthermore a preferential or exclusive formation and membrane-targeting of heteromeric (Kir3.1–Kir3.4(2)) complexes. Clear evidence for such a stoichiometric translation and channel assembly thus far is lacking.

In case of the embryonic chick heart, the subunit composition has not been studied yet. In the embryonic mouse heart, Kir3 subunit composition has been shown to undergo developmental changes (Fleischmann et al. 2004). These authors suggest a developmental increase in homomeric Kir3.4 channel from an early to the late embryonic stage. Interestingly, in adult mouse atrial myocytes the amount of Kir3.1 transcripts exceeds that of Kir3.4 transcripts by a factor of about 20 (Marionneau et al. 2005). In rat atrial myocytes, we found a ratio of about 10 (authors' unpublished observation). It is conceivable, therefore, that in the native system a higher expression level of Kir3.1 as compared with Kir3.4 is required to ensure preferential or exclusive formation of Kir3.1-containing heterotetramers.

Potential pathophysiological significance

Recently it has been shown that chronic atrial fibrillation (cAF) can be associated with changes in the properties of Kir3.x current in atrial myocytes. Dobrev et al. (2005) found a constitutive inward-rectifying current in atrial myocytes from patients with chronic atrial fibrillation, which was partially carried by Kir3 channels. Interestingly, this current but also IK(ACh) in cAF myocytes showed an about tenfold higher sensitivity to tertiapin as compared with IK(ACh) in myocytes from donors in sinus rhythm. Cha et al. (2006) demonstrated a T-Q-sensitive constitutive inward-rectifying current in atrial myocytes from chronically paced dog atria. Spontaneous or induced tachyarrhythmias in these remodelled atria could be suppressed by T-Q, in line with a study by Hashimoto et al. (2006). This could mean that within the framework of electrical remodelling that leads to cAF, homotetrameric Kir3.4 channels are formed and translocated to the membrane of atrial myocytes. The inward-rectifier currents associated with cAF in both studies were small. In a microarray study on cAF-associated changes in transcripts of ion channels and transporters in human atrial myocytes, one of the most prominent changes was a reduction in Kir3.1 mRNA, whereas Kir3.4 was not affected (Gaborit et al. 2005). In atrial tissue from patients with paroxysmal and persistent AF, Brundel et al. (2001) found a reduction in Kir3.1 protein but also a slight reduction of Kir3.4 transcripts, as compared with tissue from donors in sinus rhythm. It is conceivable that a reduction in Kir3.1 leads to formation and membrane translocation of Kir3.4 homotetramers, which is suppressed by the higher abundance of Kir3.1 over Kir3.4 under physiological conditions (Marionneau et al. 2005).

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix
  • Bender K, Wellner-Kienitz M-C, Bösche LI, Rinne A, Beckmann C & Pott L (2004). Acute desensitization of GIRK current in rat atrial myocytes is related to K+ current flow. J Physiol 561, 471483.
  • Bender K, Wellner-Kienitz M-C, Inanobe A, Meyer T, Kurachi Y & Pott L (2001). Overexpression of monomeric and multimeric GIRK4 subunits in rat atrial myocytes removes fast desentitization and reduces inward rectification of muscarinic K+ current (IK(ACh)). J Biol Chem 276, 2887328880.
  • Bender K, Wellner-Kienitz M-C & Pott L (2002). Transfection of a phosphatidyl-4-phosphate 5-kinase gene into rat atrial myocytes removes inhibition of GIRK current by endothelin and α-adrenergic agonists. FEBS Lett 529, 356360.
  • Bettahi I, Marker CL, Roman MI & Wickman K (2002). Contribution of the Kir3.1 subunit to the muscarinic-gated atrial potassium channel IKACh. J Biol Chem 277, 4828248288.
  • Brundel BJ, Van Gelder IC, Henning RH, Tuinenburg AE, Wietses M, Grandjean JG, Wilde AA, Van Gilst WH & Crijns HJ (2001). Alterations in potassium channel gene expression in atria of patients with persistent and paroxysmal atrial fibrillation: differential regulation of protein and mRNA levels of K+ channels. J Am Coll Cardiol 37, 926932.
  • Bünemann M, Brandts B & Pott L (1997). In vivo downregulation of M2 receptors revealed by measurement of muscarinic K+ current in cultured guinea-pig atrial myocytes. J Physiol 501, 549554.
  • Bünemann M, Frank M & Lohse MJ (2003). Gi protein activation in intact cells involves subunit rearrangement rather than dissociation. Proc Natl Acad Sci U S A 100, 1607716082.
  • Bünemann M, Liliom K, Brandts B, Pott L, Tseng J-L, Desiderio GM, Sun G, Miller D & Tigyi G (1996). A novel receptor with high affinity for lysosphingomyelin and sphingosine 1-phosphate in atrial myocytes. EMBO J 15, 55275534.
  • Bünemann M & Pott L (1995). Down-regulation of A1 adenosine receptors coupled to muscarinic K+ current in cultured guinea-pig atrial myocytes. J Physiol 482, 8192.
  • Cha TJ, Ehrlich JR, Chartier D, Qi XY, Xiao L & Nattel S (2006). Kir3-based inward rectifier potassium current: potential role in atrial tachycardia remodeling effects on atrial repolarization and arrhythmias. Circulation 113, 17301737.
  • Cho H, Lee D, Lee SH & Ho WK (2005). Receptor-induced depletion of phosphatidylinositol 4,5-bisphosphate inhibits inwardly rectifying K+ channels in a receptor-specific manner. Proc Natl Acad Sci U S A 102, 46434648.
  • Cho H, Nam G-B, Lee SH, Earm YE & Ho W-K (2001). Phosphatidylinositol 4,5-bisphosphate is acting as a signal molecule in α1-adrenergic pathway via the modulation of acetylcholine-activated K+ channels in mouse atrial myocytes. J Biol Chem 276, 159164.
  • Corey S & Clapham DE (1998). Identification of native atrial G-protein-regulated inwardly rectifying K+ (GIRK4) channel homomultimers. J Biol Chem 273, 2749927504.
  • Dascal N (2001). Ion-channel regulation by G proteins. Trends Endocrinol Metab 12, 391398.
  • Dobrev D, Friedrich A, Voigt N, Jost N, Wettwer E, Christ T, Knaut M & Ravens U (2005). The G protein-gated potassium current I(K,ACh) is constitutively active in patients with chronic atrial fibrillation. Circulation 112, 36973706.
  • Dobrzynski H, Janvier NC, Leach R, Findlay JB & Boyett MR (2002). Effects of ACh and adenosine mediated by Kir3.1 and Kir3.4 on ferret ventricular cells. Am J Physiol Heart Circ Physiol 283, H615H630.
  • Doupnik CA, Davidson N, Lester HA & Kofuji P (1997). RGS proteins reconstitute the rapid gating kinetics of Gβγ -activated inwardly rectifying K+ channels. Proc Natl Acad Sci U S A 94, 1046110466.
  • Fleischmann BK, Duan Y, Fan Y, Schoneberg T, Ehlich A, Lenka N, Viatchenko-Karpinski S, Pott L, Hescheler J & Fakler B (2004). Differential subunit composition of the G protein-activated inward-rectifier potassium channel during cardiac development. J Clin Invest 114, 9941001.
  • Fu Y, Huang X, Zhong H, Mortensen RM, D'Alecy LG & Neubig RR (2006). Endogenous RGS proteins and Gα subtypes differentially control muscarinic and adenosine-mediated chronotropic effects. Circ Res 98, 659666.
  • Gaborit N, Steenman M, Lamirault G, Le Meur N, Le Bouter S, Lande G, Leger J, Charpentier F, Christ T, Dobrev D, Escande D, Nattel S & Demolombe S (2005). Human atrial ion channel and transporter subunit gene-expression remodeling associated with valvular heart disease and atrial fibrillation. Circulation 112, 471481.
  • Hashimoto N, Yamashita T & Tsuruzoe N (2006). Tertiapin, a selective IKACh blocker, terminates atrial fibrillation with selective atrial effective refractory period prolongation. Pharmacol Res 54, 136141.
  • He TC, Zhou SB, Da Costa LT, Yu J, Kinzler KW & Vogelstein B (1998). A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 95, 25092514.
  • Ho IH & Murrell-Lagnado RD (1999). Molecular mechanism for sodium-dependent activation of G protein-gated K+ channels. J Physiol 520, 645651.
  • Huang CL, Feng S & Hilgemann DW (1998). Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gβγ. Nature 391, 803806.
  • Inanobe A, Yoshimoto Y, Horio Y, Morishige KI, Hibino H, Matsumoto S, Tokunaga Y, Maeda T, Hata Y, Takai Y & Kurachi Y (1999). Characterization of G-protein-gated K+ channels composed of Kir3.2 subunits in dopaminergic neurons of the substantia nigra. J Neurosci 19, 10061017.
  • Ishii M, Inanobe A & Kurachi Y (2002). PIP3 inhibition of RGS protein and its reversal by Ca2+/calmodulin mediate voltage-dependent control of the G protein cycle in a cardiac K+ channel. Proc Natl Acad Sci U S A 99, 43254330.
  • Ji S, John SA, Lu YJ & Weiss JN (1998). Mechanosensitivity of the cardiac muscarinic potassium channel – a novel property conferred by Kir3.4 subunit. J Biol Chem 273, 13241328.
  • Jin W & Lu Z (1998). A novel high-affinity inhibitor for inward-rectifier K+ channels. Biochemistry 37, 1329113299.
  • Jin W & Lu Z (1999). Synthesis of a stable form of tertiapin: a high-affinity inhibitor for inward-rectifier K+ channels. Biochemistry 38, 1428614293.
  • Kaupmann K, Schuler V, Mosbacher J, Bischoff S, Bittiger H, Heid J, Froestl W, Leonhard S, Pfaff T, Karschin A & Bettler B (1998). Human γ-aminobutric acid type B receptors are differentially expressed and regulate inwardly rectifying K+ channels. Proc Natl Acad Sci U S A 95, 1499114996.
  • Kennedy ME, Nemec J, Corey S, Wickman K & Clapham DE (1999). GIRK4 confers appropriate processing and cell surface localization to G-protein-gated potassium channels. J Biol Chem 274, 25712582.
  • Kitamura H, Yokoyama M, Akita H, Matsushita K, Kurachi Y & Yamada M (2000). Tertiapin potently and selectively blocks muscarinic K+ channels in rabbit cardiac myocytes. J Pharmacol Exp Ther 293, 196205.
  • Koyrakh L, Lujan R, Colon J, Karschin C, Kurachi Y, Karschin A & Wickman K (2005). Molecular and cellular diversity of neuronal G-protein-gated potassium channels. J Neurosci 25, 1146811478.
  • Kurachi Y, Nakajima T & Sugimoto T (1986). On the mechanism of activation of muscarinic K+ channels by adenosine in isolated atrial cells: Involvement of GTP-binding proteins. Pflugers Arch 407, 264274.
  • Lesage F, Guillemare E, Fink M, Duprat F, Heurteaux C, Fosset M, Romey G, Barhanin J & Lazdunski M (1995). Molecular properties of neuronal G-protein-activated inwardly rectifying K+ channels. J Biol Chem 270, 2866028667.
  • Marionneau C, Couette B, Liu J, Li H, Mangoni ME, Nargeot J, Lei M, Escande D & Demolombe S (2005). Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J Physiol 562, 223234.
  • Mechmann S & Pott L (1986). Identification of Na-Ca exchange current in single cardiac myocytes. Nature 319, 597599.
  • Meyer T, Wellner-Kienitz M-C, Biewald A, Bender K, Eickel A & Pott L (2001). Depletion of phosphatidylinositol 4,5 bisphosphate by activation of phospholipase C-coupled receptors causes slow inhibition but not desensitisation of G protein-coupled inward rectifier current K+ current in atrial myocytes. J Biol Chem 276, 56505658.
  • Petit-Jacques J, Sui JL & Logothetis DE (1999). Synergistic activation of G protein-gated inwardly rectifying potassium channels by the βγ subunits of G proteins and Na+ and Mg2+ ions. J Gen Physiol 114, 673684.
  • Rishal I, Keren-Raifman T, Yakubovich D, Ivanina T, Dessauer CW, Slepak VZ & Dascal N (2003). Na+ promotes the dissociation between GαGDP and Gβγ, activating G protein-gated K+ channels. J Biol Chem 278, 38403845.
  • Sadja R, Alagem N & Reuveny E (2002). Graded contribution of the Gβγ binding domains to GIRK channel activation. Proc Natl Acad Sci U S A 99, 1078310788.
  • Sadja R, Alagem N & Reuveny E (2003). Gating of GIRK channels: details of an intricate, membrane-delimited signaling complex. Neuron 39, 912.
  • Shankar H, Murugappan S, Kim S, Jin J, Ding Z, Wickman K & Kunapuli SP (2004). Role of G protein-gated inwardly rectifying potassium channels in P2Y12 receptor-mediated platelet functional responses. Blood 104, 13351343.
  • Stanfield PR, Nakajima S & Nakajima Y (2002). Constitutively active and G-protein coupled inward rectifier K+ channels: Kir2.0 and Kir3.0. Rev Physiol Biochem Pharmacol 145, 47179.
  • Sui JL, Chan KW & Logothetis DE (1996). Na+ activation of the muscarinic K+ channel by a G-protein-independent mechanism. J Gen Physiol 108, 381391.
  • Sui JL, Petit-Jacques J & Logothetis DE (1998). Activation of the atrial KACh channel by the βγ subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidylinositol phosphates. Proc Natl Acad Sci U S A 95, 13071312.
  • Takano M & Kuratomi S (2003). Regulation of cardiac inwardly rectifying potassium channels by membrane lipid metabolism. Prog Biophys Mol Biol 81, 6779.
  • Wellner-Kienitz M-C, Bender K & Pott L (2001). Overexpression of β1 and β2 adrenergic receptors in rat atrial myocytes. Differential coupling to G protein-gated inward rectifier K+ channels via Gs and Gi/o. J Biol Chem 276, 3734737354.
  • Wellner-Kienitz M-C, Bender K, Rinne A & Pott L (2004). Voltage dependence of ATP-dependent K+ current in rat cardiac myocytes is affected by IK1 and IK(ACh). J Physiol 561, 459469.
  • Yakubovich D, Rishal I & Dascal N (2005). Kinetic modeling of Na+-induced, Gβγ-dependent activation of G protein-gated K+ channels. J Mol Neurosci 25, 719.
  • Zhang H, He C, Yan X, Mirshahi T & Logothetis DE (1999). Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions. Nature Cell Biol 1, 183188.

Appendix

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Acknowledgements

The authors thank Anke Galhoff, Bing Liu and Gabriele Reimus for unfailing technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, FoRUM and the International Graduate School of Neuroscience NRW (A.R. and M.T.).

Author's current address

A. Rinne: Department of Physiology, Stritch School of Medicine, Loyola University, Chicago, USA.