小鼠胚胎心肌细胞的分离及电生理特性

本文旨在探索小鼠胚胎心肌细胞的分离方法并观察其电生理特性。应用胶原酶B消化法获得不同时期单个小鼠胚胎心肌细胞;利用全细胞膜片钳技术,记录胚胎心肌细胞的超极化激活的非选择性内向阳离子电流(If)和L-钙电流(ICa-L),并用电流钳记录其自发性动作电位。胚胎心肌细胞通过相差显微镜依据其形态和自发性收缩进行鉴定。本法分离所获得的胚胎心肌细胞容易进行全细胞膜片钳记录,可用于记录If,ICa-L．电流和自发性动作电位,己证实胚胎心肌细胞If和Ica-L的电生理特性与成年起搏细胞或心肌细胞相似。本实验建立的分离方法简单、稳定、有效、可靠,最早可获得8．5d的胚胎心肌细胞。胚胎心肌细胞的电生理记录为探索胚胎心肌细胞的电生理特性提供了一个可用的模型,并可能为某些心脏疾病产生的机制提供实验依据。     

Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34(+) cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34(+) cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP(+) cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca(2+)] ([Ca(2+)](i)) homeostasis by [Ca(2+)](i) imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP(+) cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca(2+)](i) transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca(2+)](i) oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34(+) cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.     

Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals

Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs) are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the pan-retinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, but not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiological studies indicated that with 64.7 ± 0.88% (mean ±s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca(2+) sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin and retinoid signals.     

Identification and Purification of Human Induced Pluripotent Stem Cell-Derived Atrial-Like Cardiomyocytes Based on Sarcolipin Expression

The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC) reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN). When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.     

GATA-4 induces changes in electrophysiological properties of rat mesenchymal stem cells

Background

Transplanted mesenchymal stem cells (MSC) can differentiate into cardiac cells that have the potential to contribute to heart repair following ischemic injury. Overexpression of GATA-4 can significantly increase differentiation of MSC into cardiomyocytes (CM). However, the specific impact of GATA-4 overexpression on the electrophysiological properties of MSC-derived CM has not been well documented.

Methods

Adult rat bone marrow MSC were retrovirally transduced with GATA-4 (MSC^GATA-4) and GFP (MSC^Null) and subsequently co-cultured with neonatal rat ventricular cardiomyocytes (CM). Electrophysiological properties and mRNA levels of ion channels were assessed in MSC using patch-clamp technology and real-time PCR.

Results

MSC^GATA-4 exhibited higher levels of the TTX-sensitive Na⁺ current (I_Na.TTX), L-type calcium current (I_Ca.L), transient outward K⁺ current (I_to), delayed rectifier K⁺ current (IK_DR) and inwardly rectifying K⁺ current (I_K1) channel activities reflective of electrophysiological characteristics of CM. Real-time PCR analyses showed that MSC^GATA-4 exhibited upregulated mRNA levels of Kv1.2, Kv2.1, SCN2a1, CCHL2a, KV1.4 and Kir1.1 channels versus MSC^Null. Interestingly, MSC^GATA-4 treated with IGF-1 neutralizing antibodies resulted in a significant decrease in Kir1.1, Kv2.1, KV1.4, CCHL2a and SCN2a1 channel mRNA expression. Similarly, MSC^GATA-4 treated with VEGF neutralizing antibodies also resulted in an attenuated expression of Kv2.1, Kv1.2, Kv1.4, Kir1.1, CCHL2a and SCN2a1 channel mRNAs.

Conclusions

GATA-4 overexpression increases I_to, IK_DR, I_K1, I_Na.TTX and I_Ca.L currents in MSC. Cytokine (VGEF and IGF-1) release from GATA-4 overexpressing MSC can partially account for the upregulated ion channel mRNA expression.

General significance

Our results highlight the ability of GATA4 to boost the cardiac electrophysiological potential of MSC.     

Heteromeric assembly of inward rectifier channel subunit Kir2.1 with Kir3.1 and with Kir3.4

Heteromultimerization of different pore-forming subunits is known to contribute to the diversity of inward rectifier K⁺ channels. We examined if the subunits belonging to different subfamilies Kir2 and Kir3 can co-assemble to form heteromultimers in heterologous expression systems. We observed co-immunoprecipitation of Kir2.1 and Kir3.1 as well as Kir2.1 and Kir3.4 in HEK293T cells. Furthermore, analyses of subcellular localization using confocal microscopy revealed that co-expression of Kir2.1 promoted the cell surface localization of Kir3.1 and Kir3.4 in HEK293T cells. In electrophysiological experiments, co-expression of Kir2.1 with Kir3.1 and/or Kir3.4 in Xenopus oocytes and HEK293T cells did not yield currents with distinguishable features. However, co-expression of a dominant-negative Kir2.1 with the wild-type Kir3.1/3.4 decreased the Kir3.1/3.4 current amplitude in Xenopus oocytes. The results indicate that Kir2.1 is capable of forming heteromultimeric channels with Kir3.1 and with Kir3.4.     

胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展

胚胎干细胞（embryonic stem cells,ESCs）具有自我更新、无限增殖和多向分化的特性,包括分化成心脏组织的多种类型细胞。经体细胞重编程产生的诱导多能干细胞（induced pluripotent stem cells,iPS）也被证明有类似胚胎干细胞的特性。但这些多能干细胞向心肌细胞自发分化的效率非常低,因此,如何有效地诱导这些多能干细胞向心肌细胞的定向分化对深入认识心肌发生发育的关键调控机制和实现其在药物发现和再生医学,如心肌梗塞、心力衰竭的细胞治疗以及心肌组织工程中的应用均具有非常重要的意义。该文重点综述了近年来胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展,并探讨了这一研究领域亟待解决的关键问题和这些多能干细胞的应用前景。     

High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents

Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes; however, the electrophysiological properties of hiPSC-derived cardiomyocytes have yet to be fully characterized. We performed detailed electrophysiological characterization of highly pure hiPSC-derived cardiomyocytes. Action potentials (APs) were recorded from spontaneously beating cardiomyocytes using a perforated patch method and had atrial-, nodal-, and ventricular-like properties. Ventricular-like APs were more common and had maximum diastolic potentials close to those of human cardiac myocytes, AP durations were within the range of the normal human electrocardiographic QT interval, and APs showed expected sensitivity to multiple drugs (tetrodotoxin, nifedipine, and E4031). Early afterdepolarizations (EADs) were induced with E4031 and were bradycardia dependent, and EAD peak voltage varied inversely with the EAD take-off potential. Gating properties of seven ionic currents were studied including sodium (I(Na)), L-type calcium (I(Ca)), hyperpolarization-activated pacemaker (I(f)), transient outward potassium (I(to)), inward rectifier potassium (I(K1)), and the rapidly and slowly activating components of delayed rectifier potassium (I(Kr) and I(Ks), respectively) current. The high purity and large cell numbers also enabled automated patch-clamp analysis. We conclude that these hiPSC-derived cardiomyocytes have ionic currents and channel gating properties underlying their APs and EADs that are quantitatively similar to those reported for human cardiac myocytes. These hiPSC-derived cardiomyocytes have the added advantage that they can be used in high-throughput assays, and they have the potential to impact multiple areas of cardiovascular research and therapeutic applications.     

恒流灌注分离心肌细胞及膜片钳记录钾电流方法学探讨

目的探讨膜片钳实验中豚鼠、大鼠心肌细胞急性分离过程中的观察指标和影响因素,并进行膜片钳记录。方法使用改进的Langendorff恒流灌注法分离心肌细胞和全细胞膜片钳记录钾通道电流。结果分离即刻,存活细胞可达80%以上,复钙后约40%～50%呈静息状态,余出现自发性收缩,存活细胞能够进行膜片钳封接,于室温KB液中可成活8 h以上。结论使用恒流灌注法,观测灌注压的变化,可以较客观的掌握酶解消化的程度,利于获取生理状态良好的心肌细胞。且细胞能够进行膜片钳实验。     

Hydrogen Sulfide Suppresses Outward Rectifier Potassium Currents in Human Pluripotent Stem Cell-Derived Cardiomyocytes

Aim

Hydrogen sulfide (H₂S) is a promising cardioprotective agent and a potential modulator of cardiac ion currents. Yet its cardiac effects on humans are poorly understood due to lack of functional cardiomyocytes. This study investigates electrophysiological responses of human pluripotent stem cells (hPSCs) derived cardiomyocytes towards H₂S.

Methods and Results

Cardiomyocytes of ventricular, atrial and nodal subtypes differentiated from H9 embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were electrophysiologically characterized. The effect of NaHS, a donor of H₂S, on action potential (AP), outward rectifier potassium currents (I _Ks and I _Kr), L-type Ca²⁺ currents (I _CaL) and hyperpolarization-activated inward current (I _f) were determined by patch-clamp electrophysiology and confocal calcium imaging. In a concentration-dependent manner, NaHS (100 to 300 µM) consistently altered the action potential properties including prolonging action potential duration (APD) and slowing down contracting rates of ventricular-and atrial-like cardiomyocytes derived from both hESCs and hiPSCs. Moreover, inhibitions of slow and rapid I _K (I _Ks and I _Kr), I _CaL and I _f were found in NaHS treated cardiomyocytes and it could collectively contribute to the remodeling of AP properties.

Conclusions

This is the first demonstration of effects of H₂S on cardiac electrophysiology of human ventricular-like, atrial-like and nodal-like cardiomyocytes. It reaffirmed the inhibitory effect of H₂S on I _CaL and revealed additional novel inhibitory effects on I _f, I _Ks and I _Kr currents in human cardiomyocytes.     

Mononucleated and binucleated cardiomyocytes in left atrium and pulmonary vein have different electrical activity and calcium dynamics

Cardiomyocytes consist of single- and bi-nucleus myocytes. However, the electrophysiological characteristics of mononucleated and binucleated myocytes have never been elucidated. Left atrium (LA) and pulmonary veins (PVs) are important substrate and initiators of atrial fibrillation. The purposes of this study were to evaluate the electrical properties and calcium homeostasis in mononucleated and binucleated cardiomyocytes in the LA and PVs. A whole-cell clamp, fluo-4 fluorescence, and immunocytostaining were used to investigated mononucleated and binucleated cardiomyocytes in the LA and PVs. Both mononucleated PV and LA cardiomyocytes had more positive resting membrane potential than respective PV and LA binucleated cardiomyocytes. Additionally, mononucleated PV cardiomyocytes (n = 36) had faster beating rates (2.1 ± 0.2 Hz versus 1.0 ± 0.2 Hz, P < 0.05) than binucleated (n = 10) PV cardiomyocytes. The PV (n = 18) and LA (n = 15) mononucleated cardiomyocytes had larger [Ca2?](i) transients (F/F? 1.64 ± 0.09 versus 1.20 ± 0.03 IU, P < 0.05; 1.52 ± 0.06 versus 1.19 ± 0.05 IU, P < 0.05) than the binucleated PV (n = 10) and LA (n = 10) cardiomyocytes. The immunostaining showed that mononucleated cardiomyocytes had lower Kir 2.3 and higher ryanodine receptor densities than did binucleated cardiomyocytes both in the PV and LA. In conclusions, mononucleated PV and LA cardiomyocytes contain distinctive electrophysiological characteristics with a higher arrhythmogenic activity, which indicates that cell nucleus number may potentially determine the electrical activity and calcium handling in cardiomyocytes.     

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.     

Involvement of Kv1.1 and Nav1.5 in proliferation of gastric epithelial cells

In the present study, patch clamp experiments demonstrated the expression of multiple ionic currents, including a Ba2+-sensitive inward rectifier K+ current (IKir), a 4-aminopyridine- (4-AP) sensitive delayed rectifier K+ current (IKDR), and a nifedipine-sensitive, tetrodotoxin-resistant inward Na+ current (INa.TTXR) in the non-transformed rat gastric epithelial cell line RGM-1. RT-PCR revealed molecular identities of mRNAs for the functional ionic currents, including Kir1.2 for IKir, Kv1.1, Kv1.6, and Kv2.1 for IKDR, and Nav1.5 for INa.TTXR. Pharmacologic blockade of Kv and Nav, but not Kir, suppressed RGM-1 cell proliferation. To further elucidate which subtypes of the ion channels were involved in cell proliferation, RNA interference was employed to knockdown specific gene expression. Downregulation of Kv1.1 or Nav1.5 by RNA interference suppressed RGM-1 cell proliferation. To conclude, our study is the first to delineate the expression of ion channels and their functions as growth modulators in gastric epithelial cells.     

The Interaction of Caveolin 3 Protein with the Potassium Inward Rectifier Channel Kir2.1: PHYSIOLOGY AND PATHOLOGY RELATED TO LONG QT SYNDROME 9 (LQT9)*

Mutations in CAV3 cause LQT syndrome 9 (LQT9). A previously reported LQT9 patient had prominent U waves on ECG, a feature that has been correlated with Kir2.1 loss of function. Our objective was to determine whether caveolin 3 (Cav3) associates with Kir2.1 and whether LQT9-associated CAV3 mutations affect the biophysical properties of Kir2.1. Kir2.1 current (I_K1) density was measured using the whole-cell voltage clamp technique. WT-Cav3 did not affect I_K1. However, F97C-Cav3 and T78M-Cav3 decreased I_K1 density significantly by ∼60%, and P104L-Cav3 decreased I_K1 density significantly by ∼30% at −60 mV. Immunostained rat heart cryosections and HEK293 cells cotransfected with Kir2.1 and WT-Cav3 both demonstrated colocalization of Kir2.1 and WT-Cav3 by confocal imaging. Cav3 coimmunoprecipitated with Kir2.1 in human ventricular myocytes and in heterologous expression systems. Additionally, FRET efficiency was highly specific, with a molecular distance of 5.6 ± 0.4 nm, indicating close protein location. Colocalization experiments found that Cav3 and Kir2.1 accumulated in the Golgi compartment. On-cell Western blot analysis showed decreased Kir2.1 cell surface expression by 60% when expressed with F97C-Cav3 and by 20% when expressed with P104L-Cav3 compared with WT-Cav3. This is the first report of an association between Cav3 and Kir2.1. The Cav3 mutations F97C-Cav3, P104L-Cav3, and T78M-Cav3 decreased I_K1 density significantly. This effect was related to a reduced cell surface expression of Kir2.1. Kir2.1 loss of function is additive to the increase described previously in late I_Na, prolonging repolarization and leading to arrhythmia generation in Cav3-mediated LQT9.     

Properties and functions of KATP during mouse perinatal development

BackgroundPrevailing data suggest that ATP-sensitive potassium channels (K_ATP) contribute to a surprising resistance to hypoxia in mammalian embryos, thus we aimed to characterize the developmental changes of K_ATP channels in murine fetal ventricular cardiomyocytes.MethodsPatch clamp was applied to investigate the functions of K_ATP. RT-PCR, Western blot were used to further characterize the molecular properties of K_ATP channels.ResultsSimilar K_ATP current density was detected in ventricular cardiomyocytes of late development stage (LDS) and early development stage (EDS). Molecular–biological study revealed the upregulation of Kir6.1/SUR2A in membrane and Kir6.2 remained constant during development. Kir6.1, Kir6.2, and SUR1 were detectable in the mitochondria without marked difference between EDS and LDS. Acute hypoxia–ischemia led to cessation of APs in 62.5% of tested EDS cells and no APs cessation was observed in LDS cells. SarcK_ATP blocker glibenclamide rescued 47% of EDS cells but converted 42.8% of LDS cells to APs cessations under hypoxia-ischemic condition. MitoK_ATP blocker 5-HD did not significantly influence the response to acute hypoxia–ischemia at either EDS or LDS. In summary, sarcK_ATP played distinct functional roles under acute hypoxia-ischemic condition in EDS and LDS fetal ventricular cardiomyocytes, with developmental changes in sarcK_ATP subunits. MitoK_ATP were not significantly involved in the response of fetal cardiomyocytes to acute hypoxia–ischemia and no developmental changes of K_ATP subunits were found in mitochondria.     

Assembly limits the pharmacological complexity of ATP-sensitive potassium channels

ATP-sensitive potassium channels (K(ATP) channels) are formed from an octameric complex of an inwardly rectifying K(+) channel (Kir6.1, Kir6.2) and a sulfonylurea receptor (SUR1, SUR2A, and SUR2B). In this study we have attempted to address the question of whether SUR heteromultimers can form using a combination of biochemical and electrophysiological approaches. We have constructed monoclonal stable lines in HEK293 cells co-expressing Kir6.2 with SUR1 and SUR2A. Using coimmunoprecipitation analysis with SUR isotype-specific antibodies two biochemical populations are distinguished, one containing SUR1 and the other SUR2A. It is not possible to detect immune complexes containing both SUR1 and SUR2A. Functional studies were undertaken and whole cell membrane currents were studied using the patch clamp. Concentrations of sulfonylureas and potassium channel openers were determined that selectively inhibited or activated SUR1/Kir6.2 and SUR2A/Kir6.2. In the cell line expressing SUR1/SUR2AKir6.2 we were unable to demonstrate a population of channels with unique pharmacological properties. Thus we conclude from these studies that heteromultimeric channel complexes containing both SUR1 and SUR2A are not formed, suggesting an incompatibility between different SUR subtypes. This incompatibility limits the pharmacological complexity of K(ATP) channels that may be observed in native tissues.     

Generation and Characterization of Functional Cardiomyocytes Derived from Human T Cell-Derived Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have been proposed as novel cell sources for genetic disease models and revolutionary clinical therapies. Accordingly, human iPSC-derived cardiomyocytes are potential cell sources for cardiomyocyte transplantation therapy. We previously developed a novel generation method for human peripheral T cell-derived iPSCs (TiPSCs) that uses a minimally invasive approach to obtain patient cells. However, it remained unknown whether TiPSCs with genomic rearrangements in the T cell receptor (TCR) gene could differentiate into functional cardiomyocyte in vitro. To address this issue, we investigated the morphology, gene expression pattern, and electrophysiological properties of TiPSC-derived cardiomyocytes differentiated by floating culture. RT-PCR analysis and immunohistochemistry showed that the TiPSC-derived cardiomyocytes properly express cardiomyocyte markers and ion channels, and show the typical cardiomyocyte morphology. Multiple electrode arrays with application of ion channel inhibitors also revealed normal electrophysiological responses in the TiPSC-derived cardiomyocytes in terms of beating rate and the field potential waveform. In this report, we showed that TiPSCs successfully differentiated into cardiomyocytes with morphology, gene expression patterns, and electrophysiological features typical of native cardiomyocytes. TiPSCs-derived cardiomyocytes obtained from patients by a minimally invasive technique could therefore become disease models for understanding the mechanisms of cardiac disease and cell sources for revolutionary cardiomyocyte therapies.     

Evidence against functional interaction between aquaporin-4 water channels and Kir4.1 potassium channels in retinal Müller cells

Indirect evidence suggests that the Müller/glial cell water channel aquaporin-4 (AQP4) modulates K(+) channel function of the closely associated Kir4.1 protein. We used patch clamp to compare Kir4.1 K(+) channel function in freshly isolated Müller cells from retinas of wild-type (+/+) and AQP4 knock-out (-/-) mice. Immunocytochemistry showed a comparable Kir4.1 protein expression pattern in Müller cells from +/+ and -/- retinas, with greatest expression at their end feet. Osmotic water permeability was >4-fold reduced in -/- than in +/+ Müller cells. Resting membrane potential did not differ significantly in +/+ versus -/- Müller cells (-64 +/- 1 versus -64 +/- 1 mV, S.E., n = 24). Whole-cell K(+) currents recorded with a micropipette inserted into the cell soma were Ba(2+)-sensitive and showed no significant differences in magnitude in +/+ versus -/- Müller cells (1.3 +/- 0.1 versus 1.2 +/- 0.1 nA at -160 mV) or in inwardly rectifying current-voltage relationships. Spatially resolved K(+) currents generated by pulsed K(+) injections along Müller cell bodies were also comparable in +/+ versus -/- Müller cells. Single-channel cell-attached patch clamp showed comparable unitary conductance, current-voltage data, and open probability in +/+ versus -/- Müller cells. Thus, contrary to the generally accepted view, our results provide direct evidence against functionally significant AQP4 modulation of Müller cell Kir4.1 K(+) channel function.