Reconstitution of ionic channels from human heart

J Mol Cell Cardiol21,3

15-322 (1989)

Reconstitution Joseph

of Ionic

A. Hill,

Jr., Roberto

Channels Coronado’

from

Human

and Harold

Heart

C. Strauss*

Departments of Medicine and Pharmacology, Duke University Medical Center, Durham, NC and 1 Department of Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, TX, USA (Received 26January 1988, accepted in revisedform I December 1988) J A. HILL, R. CORONADOAND H. C. STRAUSS.Reconstitution of Ionic Channels from Human Heart. Journal of Molecular and Cellular Cardiology ( 1989) 21, 315-322. This report is the first description of single ion channels from human myocardium. Using explanted human left ventricular tissue, we have studied three K-conducting and two anion-conducting channels. We report our observations of gating and ionic selectivity properties of these channels which, we argue, derive from both sarcolemmal and sarcoplasmic reticulum membranes. We postulate that one channel is the K-conducting channel (SR K channel) from human cardiac sarcoplasmic reticulum. Another channel is similar to a dimeric Cl- channel from Torpedo electroplax, displaying two equally spaced levels ofopen state conductance. KEY WORDS: K-conducting

channels; Anion-conducting

Illtroduction The heart is notorious for the geometric and temporal complexity of its membrane current events (Johnson and Lieberman, 1971; Attwell and Cohen, 1977; Noble, 1979, 1984). As such, it has been difficult to dissect the membrane currents underlying such complicated phenomena as repolarization and certain arrhythmias. There are a number of factors that contribute to this complexity, including the presence of tortuous membrane clefts that can lead to ionic accumulation and depletion artifacts (e.g. Kline and Morad, 1976; Kunze, 1977), ionic currents that overlap in time, and transmembrane currents mediated by electrogenic transport processes (e.g. ionic pumps and exchangers). In addition, electrical activity in nearly inaccessible sarcoplasmic reticulum (SR) membranes is of considerable interest (for a review, see Cailli: et al., 1985). At present, we have few specific pharmacologic tools with which to characterize these membrane events. Thus, the complete elucidation of important cardiac elec: trical processes awaits the development of new technologies in drastically simpler systems.

channels; Ion selectivity; Gating

In recent years, the single channel recording techniques have been applied to cardiac tissue, providing detailed information concerning the molecular events that underlie cardiac electrophysiology. These experimental approaches allow direct observation of the unitary current pulses that occur upon channel opening. An elaborate theoretical framework underlies inferences concerning gating mechanisms and ionic permeation. An important first step in the characterization. of basic electrophysiologic processes is the comparison of human tissues with those from non-human models. To date, no single channel studies have been reported describing ionic channels from human myocardium. Using explanted human ventricular muscle, we have observed three K-conducting and two Cl-conducting channels in artificial membranes. In this paper, we report observations of the gating and ionic selectivity properties of these channels. We argue that certain of these channels derive from sarcolemma and others from sarcoplasmic reticulum. Finally, we relate our findings to those described for ion channels from canine cardiac (and other) models that have been characterized more completely.

* Please address all correspondence to: Harold C. Strauss, M.D., Box 3845, Room 345, Bell Building, Duke University Medical Center, Durham, NC 27710, USA. 002?-2828/89/0303 15 + 08$03.00/0

IQ 1989 Academic Press l.imitczd

J. Hill et al.

316

Materials

and Methods

The materials and methods used during this study have been described elsewhere (Hill, 1987 ; Hill et al., 1989) with the exception that the tissue used was explanted human left ventricle. Briefly, slabs of tissue were procured at the time of cardiac transplantation. The myocytes were disrupted, and fractionated membranes were prepared as described (Hill et al., 1989). Lipid bilayers were constructed by painting a 1 : 1 (wt/wt) phospholipid mixture of phosphatidylethanolamine and phosphatidylserine (Avanti Polar Lipids, Birmingham, AL) dissolved in decane (20 mg/ml) across a 300 ,um diameter aperture separating two aqueous chambers (cis, truns). Typical bilayers exhibited 200 to 300 pF capacitance, and resistance was always greater than 100 GQ. The ventricular vesicles were incorporated into the planar membrane by adding them to the cis chamber under fusion conditions (Miller, 1978). All aqueous solutions were buffered with 10 mM histidine (pH = 7.1). All experiments were performed at room temperature (25 to 26°C) and under steady state conditions. Enzymatic assays were performed on each membrane preparation Uones and Besch, 1984). Prior to each determination, the concentration of sodium dodecylsulfate that provided maximal enzyme activity unmasking was identified (usually 0.4 to 0.45 mg/ml). Protein concentration was measured according to Lowry et al. (195 1) relative to bovine serum albumin. The cis chamber was connected to a voltage source while the tram chamber was held at virtual ground by a current-to-voltage converter circuit. Analog data were low-pass filtered (usually 100 Hz roll-off, 8-pole Bessel) and stored on FM tape. Later, the signal was sampled (at three to five times the filter rolloff) and stored on magnetic disk for digital analysis. Probability distributions were constructed from the records according to the following protocol : current amplitude histograms were calculated [e.g. Fig. 4(b)], and a mean for each of the approximately normally distributed populations of current samples was identified. Transition discriminators for channel opening and closing were assigned midway between two means. Most points on all I-V curves represent the

average of 3 to 10 amplitude calculations. All conductances were calculated from I -\ curves defined by at least three (generally 10 to 15) points. Reversal potentials were determined by interpolation using a fitted curve. Abbreviations: [xl, concentration of species x; KOAc = potassium acetate; SDS = sodium dodecylsulfate ; PO,,. = opening probability, defined as cumulative open time divided by the sum of cumulative open and closed times, O/(0 + C) ; Vhold= transmembrane holding potential (mV); X rnM AB//y rnM AB = electrolyte solution AB in cis chamber//electrolyte solution AB in trans chamber.

Results Characterizing the preparation

Each human heart was grossly abnormal on inspection and palpation. One heart was stiff and noncompliant with a hypertrophied free wall and septum. The other two tissue slabs were flabby and relatively fatty in texture and appearance. Pathologic diagnoses underlying end-stage cardiac failure are listed (Table 1). We employed ATPase assays to measure activities of standard membrane markers (Table 1). All three preparations yielded vesicles with significantly lower Na-K ATPase (NaK), Ca-K ATPase (CaK), and SDSunmasked NaK when compared with preparations from canine myocardium (Jones, 1988). In addition, we observed significant variation in enzyme activity among the three hearts. Channel inventory Cation channels

Five different channels were reconstituted in the planar membranes (Table 2). All five, with the exception of the Cl- channel with two conductance states (M, U), were observed from each of the three hearts. As illustrated in Figure 1, one channel was quite similar to the K-conducting channel of canine ventricular sarcoplasmic reticulum (SR K channel, Hill et al., 1989). The human channel exhibited a noisy subconductance state (0,) at approximately 60% of the total current (0,). Gating was slow and apparently cyclical; that is, each state (C, O,, 0,) had direct access to the other two (Fig. 1). Gating was minimally voltage-dependent with the probability of channel opening

Human TABLE

Cardiac

317

Channels

1. Enzymic membrane markers

Heart # 1 Heart #2 Heart #3

NaK

CaK

Azide

6.0

0.4

20 17 12

18 25

5.0

NaK

(SDS)

Orientation

9.4 20 37

Yield

36 8 33

14 5.0 3.5

NaK, Na-K ATPase activity, pmoles PJmg protein. hour; CaK, Ca-K ATPase activity, pmoles PJmg protein. hour; aside, azide-sensitive NaK, pmoles P,/mg protein. hour; NaK (SDS), total NaK in SDS-solubilized membrane; orientation, % vesicles right-side-out and tight; yield, mg protein/100 g heart. Pathologic tissue diagnoses: Heart # 1: coronary atherosclerosis, moderate ventricular hypertrophy and dilation, old anteroseptaf and posteroseptal infarcts, patchy interstitial and diffuse endocardial fibrosis with fatty change. Heart # 2 : coronary atherosclerosis, ventricular hypertrophy and dilation, mild interstitial fibrosis, endomyocardial fibrosis. Heart # 3 : coronary atherosclerosis s/p bypass surgery, moderate ventricular hypertrophy and dilation, healed anteroseptal and posteroseptal infarcts, moderate fibrous pericarditis.

Popen:lincreasing slightly with positive holding potentials. Single channel conductance ( 100 mM K + cis and tram) was 179 pS (0,) and 117 pS (0,). Measured under different conditions (332 mM K+ cis and 100 mM K+ tram, Table 2) the conductance was 184 pS (0,) and 109 pS (Or) suggesting that conductance had saturated under these conditions. All of these data are quite similar to those described for the canine cardiac SR K channel (Hill et al., 1989). Finally, the effects of Cs’ were also similar to those exhibited by the canine SR K channel. Namely, exposure an apparent decrease in to cs+ produced single channel conductance, mediated by a fast-block mechanism (data not shown). TABLE

A second K-conducting human channel was incorporated into bilayers (Fig. 2). This channel exhibited bursting kinetics of gating, with a long-lived closed state interspersed between bursts of activity. Single channel slope conductance (432 mM KOAc n’s/100 mM KOAc tram) was 52 pS. Preliminary observations suggest that this channel was similar to a channel from canine ventricle (unpublished observations). We also reconstituted a 15 pS Kconducting channel from the human myocardial vesicles [Fig. 3(a)]. Gating was non-bursting in nature. The current-voltage (I-V) relation measured in 300 mM KC1//200 mM KC1 was curvilinear [Fig. 3 (b)].

2. Inventory ofchannels

Selectivity pa.ttern

Conductancea

K-conducting K-conducting K-conducting anion

184,109 52 15 45

anion

(PS)

85

Conditionsd cisJ/trans

Gating kineticsb cyclical bursting non-bursting binary slow

mhi//mM -15 -34 -11 +21

332 432 300 200

K+//lOO K+//lOO K+//200 a-//100

+21

200 a-//100

K+ K+ K+ cl-

Fig. Fig. Fig. Fig.

1 2 3 4

cl-

’ Conductance: slope ofleast squares fitted line. b Gating kinetics: cyclical, each of the three states had direct access to the other two; bursting, bursts of channel activity alternate with periods ofquiescence; non-bursting, openings do not occur in bursts. ’ &: zero current reversal potential. d Conditions: electrolyte solutions cis and hnm.

318

J. Hill et al.

T2

PA

FIGURE 1. Sarcoplasmic reticulum K channel. Single channel records from human ventricular preparation (Heart # 3). Transitions (open state is shown upward) were selected for illustration purposes from a single I50 s record and are shown together. Experimental conditions: 166 mM KrSO, k//50 nnr K,SO, tranr, VhDld= + 10 mV. Amplitude = 4.6 pA (Or), 2.7 pA (Or), bandwidth = 70 Hz. Single channel conductance under these conditions was 184 pS (O,), 109 PS (Or).

Anion channels We studied two Cl-conducting channels. The kinetic behavior of one was strikingly similar to that described for lbrpedo electroplax Clchannels (for example, Miller, 1982 ; Hanke and Miller, 1983). It displayed four states : Upper (U), Middle (M), Lower (L) and

Closed [Fig. 4 (a)]. The states L and Closed were zero conductance (“closed”) states; L consists of short-lived flickers within a burst, and Closed corresponds to extended interburst periods of quiescence. The two states U and M were always equally spaced in conductance when measured under a variety of ionic

I

TSPA I

600 ms

FIGURE 2. Bursting K channel. Bursting K channel from human ventricular preparation (Heart # 3). Experimental conditions: 432 mu KOAc//lOtl rnbr KOAc, V,,,, = + 10 mV. Amplitude = 2.4 pA, open state is shown upward, bandwidth = 106 Hz. Single channel conductance under these conditions was 52 pS (data not shown).

Human

Cardiac

Channels

-I

0 500

PA

ICC0 rns

Voltage [mVl

FI(:URE 3. 15 pS K channel. (a) Unitary current. Experimental conditions: 300 mM KCl//200 mM KCI, l.‘hold= + 20 mV. Amplitude = +0.47 pA, open state is shown upward, bandwidth = 50 Hz. (b) Current-voltage curve. Slope conductance of the linear portion (positive voltages) = 15 pS.

and voltage conditions. Slope conductances were experimentally indistinguishable for the I, + M and M + U transitions, measuring 45 pS in 200 mM KCl// 100 mM KCl. The striking similarities of the M and U substates suggest that two similar processes (“pores”?) o p erate. Opening events occurred in bursts separated by periods of electrical silence (Closed state). Time-averaged probabilities of the appearance of U, M and L (ignoring the long-lived Closed state) from records such as that shown in Figure 4 (a) werch well described by a binomial distribution. That is, measuring one of the substate frequencies allowed us to predict the other two with a binomial distribution. Thus, the equilibrium dwell time probabilities for each M= as L= (1 -P)*, statr are given 2P( 1 - P), U = p2. Measured at + 60 m\‘, P = 0.65 provided an excellent description of the data [Fig. 4(b)]. Miller and coworkers {Miller, 1982; Hanke and Miller, 1983) have analyzed the Cl- channel from the electric organ of Torpedo and have postulated two

identical diffusion pathways operating in parallel and physically linked as a “doublebarrel” channel molecule. Our calculations for the human channel suggest that the two putative conduction processes operate independently within a burst, as in the channel from Torpedo. P open for the human Clchannel was voltage-dependent with channel opening occurring more readily at negative holding potentials. The single channel currentvoltage relation was curvilinear when measured in asymmetric solutions [Fig. 4(cj, closed circles]. This conduction behavior is significantly different from the linear I-V curves consistently observed for the Torpedo channel (Miller, 1982). It suggests that the conduction path(s) is/are asymmetric with respect to the transport of Cl- ions under these conditions, Alternatively, this asymmetry may be due in part to the asymmetric ionic environment we used for these measurements. In addition, there is evidence for the exis-

320

J. Hill et al.

(b)

Amplllude

histogram lu

- 0 open - I open w..

Current

(I3 pA total)

-2

open

-0

open

-I

open

-2

open

FIGURE 4. Anion-conducting channels. (a) Unitary Cl- current. Experimental conditions: 200 rn~ KCl//lOO rn~ KCI. VbDId= +60 mV, bandwidth = 50 Hz. Amplitudes = -2.0 pA (L + M), -2.0 (M + U). The short-lived flickers below L represent channel activity from a second channel present in the membrane. (b) Current amplitude histogram. Experimental conditions: same as (a). Area under each curve: left curve (0.154), middle (0.425), right (0.420). Range of abscissa = 8 pA. (c) Current-voltage curves. Experimental conditions: same as (a). Data points for two channels are shown: channel illustrated in (a) (a), a second channel similar to the one shown in (d), here conducting Cl- (0). Data shown as l represent either the M + U or L + M transition as these current amplitudes were experimentally indistinguishable. Slope conductances of the linear portions of each curve (positive voltages) : 45 pS (a), 145 pS (0). (d) Unitary acetate current. A highly conductive anion channel was reconstituted from the preparation of vesiculated human myocardial membranes. (Heart # 3). Two channels were present in this experiment, and open state current is directed downward. Experimental conditions: 440 rnM KOAc &//lo0 rnM KOAc ~rans, Vho,d= -40 mV. Amplitude = - 1.6 pA, bandwidth = 100 Hz. Single channel slope conductance under these conditions was 18 pS.

tence of two protein conformational states at both the U and L current levels. Apparently, there is only one conformational state at the M level. We base these assertions on the analysis of the transitions present in a 150 second record measured at -60 mV from a bilayer containing only a single channel. The record contained 1305 transitions between L and M and 3771 transitions between M and U. For each open state, 1 or 2 exponentials could be resolved: L (mean time constant Z: 1.4 ms, 12.0 ms), M (10.8 ms), and LJ (3.6 ms, 34.7 ms) . Finally, a second anion-conducting channel was observed with relatively slow, nonbursting gating properties. The I-V relation for this channel in Cl[Fig. 4(c), open circles] was curvilinear. We were able to record similar unitary events in KOAc [Fig.

4 (d)], which we interpreted to be from the same channel. Single channel slope conductance was 18 pS (440 mM KOAc cis//lOO mM KOAc trans).

Discussion The tissue used for this study was obtained from patients with end-stage cardiac disease. Tissue diagnoses on pathological examination of the specimens revealed interstitial fibrosis and fatty infiltration. All three hearts revealed changes consistent with severe atherosclerotic disease. It is possible that these disease processes may have perturbed the normal function of the channels we studied. However, the fact that we observed most (four out of five) of these channels in all three diseased hearts sug-

Human

Cardiac

gests that important features may not have been affected by disease. We cannot rule out the possibility that some of the channels observed may have derived from nonmyocyte membranes. However, our memmarker clearly brane measurements demonstrate that significant concentrations of myocyte sarcolemma and sarcoplasmic reticulum were present in the preparations. In any event, we are unable to speculate concerning the nature of channels in health) human cardiac tissue. Our measurements of membrane ATPase activities clearly show that the vesicles derived from human tissue were quite different from those derived from canine heart. Markers for sarcolemma (NaK, SDS-unmasked NaKi were substantially depressed. Vesicular orientation was also significantly different from the canine preparation. Markers for SR (CaKl and mitochondrial membrane (azide-sensitive NaKi. however, were unchanged. Thus, the most significant difference between the preparations was decreased sarcolemmal enrichment in the human preparation. One channel (Fig. 1) is similar in many respects to the K channel of SR canine cardiac (Hill et al., 1989) and skeletal muscle sources (Miller, 1978; Coronado et al., 1980; Labarca and Miller, 1981). Specifically, the large conductance, prominent substate, characteristically slow gating, and Cs+ blocking action strongly suggest that this channel derives from human myocardial sarcoplasmic reticulum. Thus, we propose that this channel is thr human counterpart to the SR K channel. We studied two additional K-conducting channels. One exhibited bursting gating kinetics, a second did not. We have described that channels from canine myocardium appear similar in essential respects (Hill, 1987 ; Our preliminary characterization of these two channels suggests that they may be similar to the delayed rectifier (Clapham and rectifier inward DeFelice, 1984! and I Sakrnann and Trube, 1984) channels of ventricular muscle. Similar channels have also been studied from calf cardiac sarcolemma ( Coronado and Latorre, 1982). Wu* studied a channel [Fig. 4(a)] whose properties of conduction and gating were similar to the “double-barrel” Cl- channel

Channels

‘321

from Torpedo electroplax described by Miller and coworkers (e.g. Miller, 1982; Hanke and Miller, 1983). These investigators have postulated that the ‘Torpedo channel is actually a dimeric complex, comprised of two identical protochannels. They base this assertion on the observation of equally spaced conductance substates, binomially distributed substatr probabilities as well as on other kinetic and probabilistic arguments. With the human Cl- channel, we observed two levels of open state current I,M, I_:! with transitions well described by a binomial distribution. The conductance substates gated independently during any given burst (time scale, tens of milliseconds), but were linked b> the presence of a long-lived Closed state / time scale. seconds) that punctuated each record. Thus, there were at least two closed states I,. Closed 1. Based on our observation of transitions to and from the long-lived Closed state, we can rule out the possibility that these records represent two independent channels operating simultaneously. Under no circumstance did we observe single L -+ M transitions without M -+ TJ transitions as well. Thus, the two substates M and U always appeared together and are kinetically coupled. Several features of the Cl channel from human myocardium have been described fbr the channel from Torpedo. We have demonstrated equally spaced conductances of the M and C substates as well as binomially distributed time-averaged probabilities of appearance for U, M and L. Opening probabilit) also increased with increasingly negative ~‘tlol, . These measurements are in agreement with those described for the Torpedo channel. Certain differences, however, are apparent between these channels. The conductance ot the Cl- channel from human tissue was significantly greater than that of the %rpt~dt~ channel (45 pS in 200 mM KCl//lOO mM K(I:I vs 20 pS in 150 mM Cl- I Miller, 1982). In we are unable, at present, to addition, account for the fact that two exponential5 were resolved for the U and L states. Thrsc* calculations suggest that the U and L states are each composed of two states with mean lifetimes as given. This paper is the first description of tht reconstitution of single ionic channels from

322

J. Hill

human myocardium. We have studied five different channels which we argue derive from both sarcolemmal and sarcoplasmic reticulum membranes. Each channel appears to have a counterpart that has been studied in other species either by our group or by others. Studies such as these, relating human and non-human cardiac ion channels, help to set the stage for further detailed characterization of these membrane proteins.

et al.

.hknowledgement.r We are grateful to Drs Andrew S. Wechslcr and James E. Lowe for their cooperation in providing fresh surgical specimens. We thank Joseph C. Greenfield, Jr. for his continued support. Supported by NHLBI grants HL19216 and 17670, and NIGMS 5T32 GM 07 171 and 07105. RC was supported by an Established Investigatorship from the Xmerican Heart Association.

References ATTWELL D, COHEN 1 (1977) The voltage clamp ofmulticellular preparations. Prog Biophys Molec Biol31: 201 245. CAILL~ J, ILDEFONSE M, ROUCIER 0 (1985) Excitation-contraction coupling in skeletal muscle. Prog Biophys Molrc Biol46: 1855239. CLAPHAM DE, DEFELICE LJ (1984) Voltage-activated K channels in embryonic chick heart. Biophys J 45: 4G42. CORONADOR, LATORRE R (1982) Detection of K+ and Cl- channels from calf cardiac sarcolemma in planar lipid bilayer membranes. Nature 298: 849-852. CORONADO R, ROSENBERG RL, MILLER C (1980) Ionic selectivity, saturation. and block in a K+-selective channel from sarcoplasmic reticulum. J Gen Physio176: 425-446. HANKE W, MILLER C (1983) Single chloride channels from Torpedo electroplax: activation by protons. J Gen Physiol 82 : 25-45. HILL JA (1987) Ion conduction and selectivity in a potassium channel from cardiac sarcoplasmir reticulum. Ph.D. Dissertation, Duke University. HILL JA. CORONADO R, STRAUSS HC (1989) Potassium channel of cardiac sarcoplasmir reticulum is a multi-ion channel. Biophys J 55: 35 45. JOHNSON EA, LIEBERMAN M (197 1) Heart: excitation and contraction. Annu Rev Physiol33: 479 532. JONES LR (1988) Rapid preparation of canine cardiac sarcolemmal vesicles by sucrose flotation. Methods Enzymol 157 : 85-9 1. JONES LR, BESCH HR (1984) Isolation of Canine Cardiac Sarcolemmal Vesicles. In : Methods itl Pharmacologv,edited by A Schwartz. New York, Plenum Press, pp l-12. KLINE RP, MORAD M (1976) Potassium e&x and accumulation in heart muscle. Evidence from K+ electrode experiments. Biophys J 16: 367-372. KUNZE DL (1977) Rate-dependent changes in extracellular potassium in the rabbit atrium. Circ Res 41: 122 127. LABARCA P, MILLER C (1981) A Kf-selective, three-state channel from fragmented sarcoplasmic reticulum of frog leg muscle. J Membr Biol61: 31-38. LOWRY OH, ROSEBROUGH NJ, FARR AC, RANDALL RJ (1951 J Protein measurement with the folin phenol reagents. ,J Biol Chem 193: 2655275. MILLER C (1978) Voltage-gated cation conductance channel from fragmented sarcoplasmic reticulum. Steady state electrical properties. J Membr Biol40: l-23. MILLER C (1982) Open-state substructure of single chloride channels from Torpedo electroplax. Phil Tram R Sot B Biol Sci 299: 401~411. NOBLE D (1979) The Initiation ofthc Hmrtbeat. New York, Oxford University Press. NOBLE D (1984) The surprising heart: A review of recent progress in cardiac electrophysiology. J Physiol iLond) 353: I- 50. SAKMANNB, TRUBE G (1984) Conductance properties of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart. J Physiol (Lond) 347: 641-657.