0031-3998/ 92/ 3202-0230$03.00/0 PEDIATRI C RESEARCH Vol. 32, No. 2, 1992 Copyright © 1992 International Pediatric Research Foundation, Inc. Printed in U.S. A. ATP-Sensitive Potassium Channels in Neonatal and Adult Rabbit Ventricular Myocytes FUHUA CHEN, GLENN T. WETZEL, WILLIAM F. FRIEDMAN, AND THOMAS S. KLITZNER Departm ent a/Pediatrics, Division a/ Cardiology, University a/California at Los Angeles. School a/Medicine, Los Angeles. California 90024 ABSTRACT. The properties of the ATP-sensitive potas­ metabolic inhibition, large K+ currents develop and the K+ sium (KATP) current were studied in freshly isolated rabbit current-voltage relation may become almost linear (3, 4). Und er ventricular myocytes using the patch clamp technique. these conditions, increased outward current results in action Removing ATP from the bath (intracellular) solution acti­ potential shortening, thereby decreasing Ca2+ influx and devel­ vated a large K+ conductance in patches from neonatal oped tension. In addition, the increase in extracellular K+ may cells with properties similar to those of KATP channels in result in a dispersion of refractoriness, precipitating arrhythmias other preparations. In membrane patches from neonatal and abnormal contractions during hypoxia or ischemia (5). ventricular myocytes, the density of KATP channels was Recent studies suggest that increased K+ current in hypoxic higher than the density of inwardly rectifying K+ channels and ischemic myocardium is related to the presence of KAT? and the mean patch KATP current was approximately 10 channels that are activated when internal ATP levels fall (4, 6, times that of the inwardly rectifying K+current, at a patch 7). KAT? channels have also been identified by single-channel membrane potential of -60 mV. Glibenclamide (10 recordin g techniques in cells from other tissues (8). in the bath solution decreased the number of functional Although the KAT? channel has been extensively investigated KATP channels, the open-state probability, and the mean in a variety of adult cardiac myocytes, information regarding the patch membrane current. The single-channel conductance characteristics of the channel in immature mammalian heart is of the KATP channel was dependent on the external K+ limited to cultured rat ventricularcells (9). Further, the properties concentration, and the relationship between channel con­ of the channel have not been compared between neonatal and ductance and external K+ concentration was fit by an adult myocytes. Using the patch clamp technique for single­ exponential equation. In addition, the voltage dependence, channel recording, we have characterized the single-channel con­ channel density, and open-state probability of this channel ductance properties, channel density, and open-state probability were compared between neonatal and adult isolated ven­ of this channel in the freshly isolated neonatal rabbit ventricular tricular myocytes. The single-channel conductance and myocytes. In addition, the effects of reduction in cytoplasmic channel density of the KATP channel in neonatal myocytes ATP concentration on the outward K+current and action poten­ were significantly smaller than in adult cells. These results tial duration have been investigated. Comparison of results ob­ suggest that age-related changes occur in the properties of tained from neonatal and mature myocytes suggests significant KATPchannels. (Pediatr Res 32: 230-235, 1992) differences in KAT? channe l properties between the two age groups. Abbreviations MATERIALS AND METHODS IK 1, inwardly rectifying K+ current KATPchannel, ATP-sensitive K+ channel Preparation. Isolated ventricular myocytes were obtained from HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic neon atal (2- 5 d old, mean ± SEM 3.4 ± 0.3 d, n = 16) New acid Zealand White rabbits (50-125 g, 108 ± 6.8 g, n = 14) by Popen, open-state probability enzymatic dissociation as described previously (10, I 1). In brief, Ime,n, mean patch current neonatal rabbits were anticoagulated with 1000 units of heparin pS, picosiernens and anesthetized with pentobarbital (50 mg) by intraperitoneal [K+], K+ concentration injection. The heart was rapidly excised. The aorta was cannu­ [K+]", external K+ concentration lated and perfused for 3 min at a rate of 2.5 mLjmin with Ca2+­ [K"], internal K+ concentration free Tyrode's solution contai ning (in mM) NaCl, 136; KCI, 5.4; NaH 2P04, 0.33; MgCb, 1; HEPES, 10; mannitol, 4; thiamine HCI, 0.6; glucose, 10; and pyruvic acid, 2. The perfusate was switched to Ca2+-free Tyrode's solution containing collagenase (300 UjmL, Type I; Sigma Chemical Co., St. Louis, MO) and Near the resting membrane potential (--80 mV), potassium protease (0.35 UjmL, Type XIV; Sigma Chemical Co.), which is the dominant permeant cation in ventricular myocardium (I). was recirculated with a peristaltic pump (Minipulse 2; Gilson, However, outward K+ currents become quite small in the mem ­ Middleton, WI) for 7 to 9 min. Thereafter, enzymes were washed brane potential range near the action potential plateau, due to out for 3 min with 0. 1 mM Ca2+ Tyrode's solution containing inward rectification ofthe K+current-voltage relation (2). During (in mM) NaCI, 136; KCI, 5.4; CaCb, 0.1; NaH2P04, 0.33; MgCb, Received September 23, 1991; accepted March 6, 1992. 1; HEPES, 10; mannitol, 4; thiamine HCI, 0.6; glucose, 10; and Correspondence and reprint requests: Tho mas S. KJitzner, M.D., Ph.D., De­ pyruvic acid, 2. The ventricle was opened and gently shaken in partment of Pediatrics, UCLA School of Medicine, Los Angeles, CA 90024. 0. 1 mM Ca2+ Tyrode's solution to disperse individual, relaxed Supported in part by funding from the NIH (HL-0 1347), the American Heart myocytes. Association, Greater Los Angeles Affiliate (788 IG), and the Variety Club, J.H. Nicholson Endowment. Dr. Chen is supported by a Young Investigator Award Myocytes from adult rabbits (3 to 3.5 kg) were isolated in a from the American Academy of Pediatrics, Section on Cardiology. similar fashion. After i.v. anti coagulation (heparin, 1500 U) and 230 KATP CHANNEL IN NEONATAL CARDIAC CELLS 231 sedation (pentobarbital, 150 mg), hearts were excised and per­ is well within the tolerance established for whole cell recording fused at a rate of 45 mL/min. Solution composition was identical (16). to that used for neonatal rabbits, but enzyme perfusion time was Action potential recording. Action potentials were measured in increased to 40 to 45 min. The viability ofmyocytes prepared in isolated myocytes using the patch clamp amplifier in the current this fashion and the suitability for physiologic studies has been clamp mode. After establishing electrical continuity between the discussed previously (10-12). microelectrode and the cell interior, cells were stimulated at 121 Patch clamp technique. Ventricular cellswere placed in a small min by passing lO-ms current pulses at 1.5x threshold. As for volume recording chamber (1 mL) on the stage of an inverted whole cell K+ current recording, action potential duration was microscope (Diaphot, Nikon Inc., Garden City, NY). A standard measured both upon the puncture of the cell membrane and patch clamp technique, similar to that of Hamill et al. (13), was after 20 min of dialysis of the cell interior with ATP-free pipette used and has been described previously (11). After formation of solution. Action potentials were recorded, digitized, and stored a gigaseal between the pipette tip and the surface membrane of on a computer (IBM AT) for later analysis. The bath and pipette a myocyte, a membrane patch was pulled from the cell and solution were the same as for whole cell voltage clamp experi­ currents recorded in the inside-out patch configuration. A List ments. EPC-7 patch clamp amplifier (List-Electronic, Darmstadt-Eber­ Statistical methods. Data are presented as mean ± SEM. stadt, Germany) was used to measure single channel currents Groups were compared using a two-tailed t test. A p value of < using Corning soft, thin-walled 8161 glass capillaries (Corning 0.05 was used to indicate significance. Slopes of lines were Glass Co., Horsehead, NY) with a tip resistance of 2-6 MQ calculated by linear regression. Statistical analysis was performed (typically 4 MQ) when filled with internal solution. Sealing using BMDP software (Los Angeles, CA). resistance varied from 10 to 25 GQ. Single-channel currents and membrane potential were displayed on a storage oscilloscope (5113; Tektronix, Beaverton, OR) and recorded on a Gould RESULTS chart recorder (Brush 440; Gould Inc., Cleveland, OH) or digi­ Identification 0/ the KATP channel in neonatal excised mem­ tized (1 kHz) and stored on the hard disk of a microcomputer brane patches. Figure 1 shows single-channel currents recorded (IBM AT, IBM Co., White Plains, NY), using Axolab 1100 from a typical neonatal membrane patch with [K], = [K], = 150 acquisition hardware and pCLAMP software (Axon Instruments, mM and the membrane potential held at -60 mV. With 2 mM Burlingame, CA). Currents were low-pass filtered at 100-200 Hz ATP present in the bath (intracellular) solution, no channel with an eight-pole bessel filter (902LPF; Frequency Devices Inc., openings were observed (beginning ofthe upper trace). Removing Haverhill, MA). ATP from the bath solution activated a large conductance chan­ Popen was calculated as the mean K+ channel current for a nel (Fig. 1, upper trace), which failed to open when ATP was given patch (Imean) divided by the maximum superposition num­ restored to the bathing solution. The emergence of this channel ber (N, the estimated number of active channels observed in the upon removal of ATP was reversible and reproducible (Fig. 1, patch) multiplied by the unitary current (i): lower trace). Equivalent results were recorded from a total of 14 inside-out membrane patches. These currents are similar to those Popen = Imean/(Ni) recorded from the ATP-sensitive K+ channel previously charac­ Imean was determined by subtracting the baseline current from terized in adult ventricular myocytes (6) and in other prepara­ the measured current averaged over at least lOs.