~using the patch clamp technique in the whole-cell, cell-attached and inside-out patch
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- Reynard Winfred Tate
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1 4751 Journal of Phy8iology (1996), 493.1, pp f 14; Competition between Mg2" and spermine for a cloned IRK2 channel expressed in a human cell line Takeshi Yamashita, Yoshiyuki Horio, Mitsuhiko Yamada, Naohiko Takahashi, Chikako Kondo and Yoshihisa Kurachi * Department of Pharmacology II, Faculty of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565, Japan 1. A cloned inwardly rectifying K+ channel, IRK2, was expressed in a human cell line, human embryonic kidney (HEK) 293T. Its electrophysiological properties were examined ~using the patch clamp technique in the whole-cell, cell-attached and inside-out patch configurations. 2. The cells transfected with IRK2 cdna exhibited a K+ current which showed classical properties of inwardly rectifying K+ channels at both whole-cell and single-channel levels. 3. In the inside-out patch configuration, intracellular Mg2+ (Mg2+) blocked the outward currents in a voltage-dependent and virtually time-independent manner. Mg2+ (1-100 /UM) caused a decrease in the unitary current amplitude of the IRK2 channel by inducing subconducting levels. 4. In the absence of Mg2+, intracellular spermine blocked the outwardly flowing IRK2 currents in a voltage- and time-dependent manner. Spermine (1-100 nm) did not affect the unitary channel current amplitude but reduced the channel open probability. The spermine block showed a slower time and steeper voltage dependence than the Mg2+ block. 5. When both these blockers were present, Mg2+ apparently attenuated the inhibitory effect of spermine on the outwardly flowing IRK2 currents. This interaction was voltage and time dependent, and could be well explained by a model in which Mg2+ and spermine competitively bind to the channel with their individual first-order kinetics. This competition would induce time-dependent transits of the channel between the Mg21+- and spermine-blocked states via a single open state, thereby preserving a certain size of persistent outward currents at depolarized potentials. A variety of cells possess inwardly rectifying K+ channels, through which currents flow more readily in the inward than in the outward direction. The K+ channels play a significant role in maintaining the resting membrane potential near the K+ equilibrium potential (EK), the longlasting action potential (e.g. in cardiac ventricular myocytes), and consequently controlling the cellular excitability (Hagiwara, Miyazaki & Rosenthal, 1976; Fletcher & Chiappinelli, 1992; Ishikawa, Wegman & Cook, 1993). The inwardly rectifying property of this type of K+ channel has been believed to arise from a combination of intracellular Mg2+ (Mg2+)-mediated blockade and an intrinsic voltage-dependent gating process (Kurachi, 1985; Matsuda, Saigusa & Irisawa, 1987; Matsuda, 1988; Ishihara, Mitsuiye, Noma & Takano, 1989; Stanfield et al. 1994a; Jan & Jan, 1994). The contribution of each component to the inward rectification differs among distinct types of inwardly rectifying K+ channel (Horie, Irisawa & Noma, 1987; Horie & Irisawa, 1987; Lu & MacKinnon, 1994; Taglialatela, Wible, Caporaso & rown, 1994). The rectification provoked by the Mg2+ block results mainly from induction of the subconducting levels of outwardly flowing K+ channels, while the intrinsic gating reduces the outward currents by decreasing the channel open probability at potentials more positive than EK (Kurachi, 1985; Matsuda et al. 1987; Matsuda, 1988; Ishihara et al. 1989). The gating process proceeds more slowly than the Mg1+ block. In cardiac background inwardly rectifying K+ channels, for instance, Mg"+ causes an instantaneous * To whom correspondence should be addressed.
2 144 1T Yamashita and others J Physiol inward rectification, which is followed by a time-dependent enhancement of the rectification by the intrinsic gating mechanism (Ishihara et al. 1989). Recently, it has been reported that the 'intrinsic gating' results from a voltagedependent blockade of outwardly flowing channel currents caused by intracellular polyamines, such as spermine and spermidine (Fakler, riindle, Glowatzki, Weldemann, Zenner & Ruppersberg, 1994a; Ficker, Taglialatela, Wible, Henley & rown, 1994; Lopatin, Makhina & Nichols, 1994; Yamada & Kurachi, 1995). These intracellular substances, i.e. Mg2+ and polyamines, may interact with each other on the K+ channels in provoking the physiologically observed inwardly rectifying property. Actually, it was reported that the closing of the cardiac background inwardly rectifying K+ channel by the 'intrinsic gate' is decelerated by Mg2+ (Ishihara et al. 1989). However, the mechanism of this interaction has not been yet clarified. In the present study, we examined the interaction between Mgi+ and spermine on a cloned inwardly rectifying K+ channel, IRK2, which is expressed in heart and brain (Koyama, Morishige, Takahashi, Zanelli, Fass & Kurachi, 1994; Takahashi et at. 1994). The present results demonstrate that Mg"+ and spermine compete with each other in binding to the channel. METHODS Construction of human embryonic kidney (HEK) 293T cell expression vectors The recombinant plasmid containing IRK2 cdna (Takahashi, et al. 1994) cloned from a mouse brain cdna library was digested with restriction endonucleases amhi and XhoI (both sites in luescript polylinker) to excise the entire cdna insert. This fragment was gel purified and ligated into an expression vector for mammalian cells (pcdna3; Invitrogen, CA, USA) which uses CMV (cytomegalovirus) promotor for direct expression and had been digested with amhi and Xhol. Transient expression in HEK 293T cells The IRK2-pcDNA3 plasmid was transfected into HEK 293T cells fed with Dulbecco's modified Eagle's medium (DMEM; Nikken, Kyoto, Japan) containing 10% fetal calf serum (Gibco-RL, Gaithersburg, MD, USA). Cells (7 x 104 per coverslip) were seeded onto glass coverslips (10 mm diameter) coated with poly-d-lysine (Sigma). After h, the cells were washed once with OPTI-MEM (Gibco-RL) and were transfected with the IRK2-pcDNA3 plasmid using lipofectoamine (Gibco- RL) in OPTI-MEM. Six hours after transfection, fetal calf serum was added to the medium to a concentration of 10% (v/v), and the cells were incubated for h. After this period, the cells were washed and incubated in DMEM containing 10% fetal calf serum for a further h before electrophysiological assay. Electrophysiological recordings Whole-cell and single-channel currents were measured at room temperature (-25 C) using the tight-seal patch clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The tip resistance of the electrodes was 1-3 MQl for the whole-cell clamp and 5-7 MQI for the single-channel recordings. The tips of the patch electrodes were coated with Sylgard (Dow Corning) and fire polished. Currents were measured using a patch clamp amplifier (EPC-7; List, Darmstadt, Germany), monitored with a high-gain digital storage oscilloscope (VC-6025; Hitachi, Tokyo) and recorded on videotape using a PCM converter system (VR-1O; Instrutech Corp., New York). For subsequent analysis, data were reproduced, low-pass filtered at -1 khz (-3 d) by an 8-pole essel filter (Frequency devices, Haverhill, MA, USA), sampled at 5 khz and analysed off-line on a computer (Macintosh Quadra 700, Apple Computer Inc., Cupertino, CA, USA), using commercially available software (Patch Analysit Pro; MT Corporation, Hyogo, Japan). In some experiments, data were reproduced with a filter of 3 khz and a sampling rate of 10 khz. Results were expressed as means + S.D., and significant differences between values were assessed using Student's unparied t test. P < 0 05 was considered significant. Solutions and chemicals In whole-cell clamp experiments, the bathing solution contained (mm): 140 KCl, 1-8 CaCl2, 0 53 MgCl2, 5.5 glucose, and 5.5 Hepes-KOH (ph 7 4). For desired concentrations of K+, K+ was replaced with equimolar Nae. The pipette solution contained (mm): 140 KCI, 5 K2ATP, 1 MgCl2, 5 EGTA and 5 Hepes-KOH (ph 7 3). In single-channel recordings, the bathing solution was the same as that for the whole-cell clamp experiments. The pipette solution contained (mm): 140 KCl, 1 CaCl2, 1 MgCl2, and 5 Hepes-KOH (ph 7 4). For the inside-out patch experiments, the internal solution contained (mm): 140 KCl, 5 EGTA, and 5 Hepes-KOH (ph 7 3) with variable concentrations of MgCl2 (free Mg2+ concentrations specified in Results). Mg2+-free internal solution contained (mm): 140 KCl, 5 EDTA, and 5 Hepes-KOH (ph 7 3). ATP (K+ salt) and spermine were purchased from Sigma and Wako Pure Chemical Industries, Ltd (Osaka, Japan), respectively. All the other chemicals were of the highest purity available and obtained from commercial sources. RESULTS Whole-cell and single-channel properties of IRK2 current expressed in HEK 293T cells Figure 1A illustrates a series of whole-cell current responses of HEK 293T cells evoked by +20 mv incremental voltage steps (between -100 and +40 mv) from a holding potential of 0 mv. The bathing solution contained 145 mm K+. In non-transfected cells, small leak currents were evoked by these voltage steps (Fig. 1A a). In the cells transfected with IRK2 cdna, on the other hand, a large inward and relatively small outward currents were induced by hyperpolarizing and depolarizing voltage steps, respectively (Fig. 1A b). Inactivation of the expressed current at hyperpolarized potentials varied from cell to cell. Although the cell capacitance did not differ between the non-transfected and transfected cells ( vs pf, n = 10 in both cells, not significant), the input resistance in the 145 mm K+-containing solution was larger in the non-transfected than in the transfected cells ( vs MQ, n = 10 in both cells, P < 001).
3 J Fhy8iol Mgz± and spermine competition for 1RKA channels 145 a2+ (10,M) and Cs+ (100 #M) added to the bathing solution blocked the expressed IRK2 current in a time- and voltagedependent manner (Fig. la c, d and ). Figure 1 C illustrates the current-voltage relationships of IRK2 current expressed in HEK 293T cells in the presence of various concentrations of extracellular K+ ([K+]O). The relationships were examined by a ramp pulse protocol (1 mv (10 ms)-' from -150 to +20 mv) under voltage clamp conditions. oth slope conductance and reversal potential of the expressed currents depended on [K+]0. When the slope conductance at 5 mm K+ was defined as 1, those at 20 and 80 mm K+ were 1P and (n= 5), respectively. The reversal potential of the transfected cells was reduced by mv (n = 5) per tenfold increase in [K+]0. This is a slightly weaker response than expected for a K+-selective channel and might be due to the existence of some intrinsic currents of HEK 293T cells. Single-channel currents of the expressed IRK2 channels were recorded in the cell-attached configuration (Fig. 2). Currents flowing through a single IRK2 channel were observed only at potentials more negative than EK (0 mv), thus showing a strong inwardly rectifying property (Fig. 2A). The unitary conductance of the IRK2 channel with 145 mm K+ in the pipette was ps (n = 3, Fig. 2), a value similar to that measured in Xenopus oocytes (Takahashi et at. 1994). Openings of the channel did not occur at random but in bursts. As the membrane was hyperpolarized from -10 to -100 mv, the duration of bursts decreased and long gaps between bursts appeared. The open time of the channel increased between -20 and -40 mv. It then decreased slightly with further hyperpolarization. At potentials more negative than -40 mv, the gaps between bursts became longer. These whole-cell and single-channel properties of the IRK2 current are the same as those expressed in Xenopus oocytes (Takahashi et al. 1994). lockade of outwardly flowing IRK2 currents by Mgi+ Mg2+ is known to induce the inward rectification of various inwardly rectifying K+ channels, including cardiac background inwardly rectifying K+, ATP-sensitive K+ (KATP), muscarinic K+ (KACh), and cloned IRK1 channels (Horie et al. 1987; Horie & Irisawa, 1987; Matsuda et al. 1987; Vandenburg, 1987; Matsuda, 1988; Stanfield et al. 1994b). Therefore, we examined the effects of Mg2+ on expressed IRK2 currents in the inside-out patch configuration. A a f-i-- f r b IRK2 L===P+r 10-sM al < -200 C -600 o f AGO Voltage (mv) 50 c I 1 0 #m a2' L C V (mv) /' N d 1 00 /ZM Cs+ 5mM K / / 20 mm Ko / 1I ms 1 na 80 mm Ko ; 3A I (na) Figure 1. WVhole-cell current recordings of IRK2 channels expressed in HEK 293T cells A, currents evoked by a series of 250 ms voltage steps in 20 mv increments from -100 to +40 mv. The holding potential was 0 mv. The bathing solution contained 145 mm K+. Arrows indicate the zero current level. a and b, currents in a non-transfected and an IRK2-transfected cell, respectively. c and d, the effects of external 10 um a2+ and 100 /SM Cs+, respectively, on the expressed IRK2 current., current-voltage relationships at the end of the command pulses shown in A b-d; 0, 140 K+; A, 10/M a2p;, 100 /SM Cs+. C, the effects of [K+]. concentration (5, 20 and 80 mm) on the currents of a cell transfected with IRK2. Currents were elicited by a ramp voltage pulse from -150 to +20 mv over a duration of 1'7 s. Each trace is an average of five records which were not leak subtracted.
4 140 4l' Yamashita and others J Phy.io' f Figure 3A shows the effect of Mg2+ on the outward IRK2 channel currents in a patch which probably contained three channels. When the Mg2+-free internal solution was perfused to the inside-out patch, the outward currents through IRK2 channels appeared. The outward currents during depolarizing voltage steps to +60 mv showed practically no time-dependent alterations (Fig. 3A a). Micromolar concentrations of Mg2+, added to the internal solution, blocked the outward currents (Fig. 3A b-d). At 1 /IM the outward IRK2 currents decreased time dependently during the command voltage step to +60 mv (Fig. 3A b). The subconducting levels (half of the current level of a single-channel current) were also clearly noted (Fig. 3A b, arrowheads). Higher concentrations of Mg.1 (>5/M) caused an instantaneous block and flickerings of the currents (Fig. 3A c). In the presence of 100 JSM Mg2+, only trivial outward currents were detected (Fig. 3A d). The concentration-response relationship of Mg2+ block on IRK2 channel currents was examined at various potentials in the patches containing more than ten channels in Fig. 3. The ramp pulse protocol was used. Mg2+ blocked the outward currents flowing through IRK2 channels in a concentration- and voltage-dependent manner. The concentration-response relationships at +40 mv (Fig. 3C) could be fitted by the Hill equation with an IC50 value of - 11 um and a Hill coefficient of -1. lockade of outwardly flowing IRK2 currents by intracellular spermine For IRKI and cardiac KACh channels, blockade of the outwardly flowing currents by cytoplasmic polyamines, such as spermine and spermidine, has recently been proposed as a mechanism of inward rectification, which had been recognized as an intrinsic gating (Fakler et al. 1994a; Ficker et al. 1994; Lopatin et al. 1994; Yamada & Kurachi, 1995). Figure 4 shows the effects on IRK2 channels of spermine applied to the intracellular side of patch membranes. Nanomolar concentrations of spermine blocked the outwardly flowing IRK2 channel currents at +40 mv in a concentration- and time-dependent manner (Fig. 4A). In the presence of spermine, the outward IRK2 channel openings gradually decreased during depolarizing voltage steps, while the unitary current amplitude was unaffected. As the concentration of spermine was increased, the decay of the channel openings during command steps was accelerated, which was more evident in the patches containing many channels as shown in Fig. 4. On the other hand, the inward currents at -40 mv were not significantly affected by intracellular spermine (1-100 nm). A +40 mv -1u mv -, -20 mv +&N49oo *V% - Wp "4t0 Ij p*ug -30 mvv N_W -40MV-4h,t_& A,fi.j I II J.L. 1 IAI..i1, -50 mv-i L -ja I i. JA I L -60 mv Al4Afrl IaJ I -. -[I -80 mv Jl.j Q CL e Uc.Y C c Votge(V -100 mv 2 pa 2 s Figure 2. Single-channel recordings from a cell-attached membrane patch of HEK 293T cells expressing the IRK2 channel A, current traces recorded at the membrane potentials indicated to the left of each trace. Arrows indicate the zero current level., the current-voltage relationship of the single-channel records. The mean slope conductance was -38 ps at potentials negative to -20 mv.
5 J. Physiol Mg t and spermine competition fbr IRK2 channels 147 The concentration-dependent inhibition by spermine of IRK2 currents at +40 mv measured at the end of the command steps could be fitted by the Hill equation with an IC50 value of -3 nm and a Hill coefficient of -1 8 (Fig. 4C, upper panel). Consistently, the reciprocal plot of the decay time constant against spermine concentration was fitted by a polynomial function of order (Fig. 4C, lower panel). The effect of 10 nm spermine on IRK2 current at different membrane potentials is shown in Fig. 5. The outwardly flowing IRK2 currents were more promptly blocked by spermine, as the membrane was more depolarized from EK. As a result, the current size measured at the end of command pulses became smaller as the membrane was progressively depolarized, i.e. a negative slope conductance region was formed at potentials more positive than +10 mv (Fig. 5A, upper panel; see also Fig. 7). At the potentials more negative than EK, however, little or no significant inhibition of the steady-state inward IRK2 currents was induced by spermine. The lower panels of Fig. 5A show the unblocking process of the spermineinduced block at hyperpolarized potentials. Here, hyperpolarizing voltage steps from +10 to -100 mv were applied after a conditioning pulse to +40 mv (200 ms duration) in the absence and presence of spermine (10 nm). In the absence of spermine, there was no significant timedependent alteration of inward currents during the command steps. In the presence of spermine, the current at +40 mv became negligible. The outward IRK2 current during command steps to +10 mv increased to a certain steady level in a time-dependent manner, following a small instantaneous current jump. At negative potentials, inward K+ currents increased time dependently. The rate of the current increase became faster as the membrane was more hyperpolarized. This time-dependent current alteration caused by unbinding of spermine at hyperpolarized potentials is similar to the activation gating described in cardiac background inwardly rectifying K+ channels (Kurachi, 1985; Fakler et al. 1994a; Ficker et al. 1994; Lopatin et al. 1994). A a Mg2+ free b 1 MMMg2+ C 10/uM Mg2+ V (mv) I (pa) I, Mg2' free C c 0 D 0-6 -c ai) 0-4 a1) co Er 02 d 100/,M Mg2+ J2 pa 50 ms Free Mg2+ concentration (mnol 1-') 10-2 Figure 3. The effects of Mg2+ on the expressed IRK2 channel A, various concentrations of free Mg2+ (0, 1, 10 and 100 /LM) were perfused to an inside-out patch containing three IRK2 channels. Voltage steps to +60 mv were applied from the holding potential of -60 mv. Continuous and dashed lines indicate the zero current and single-channel levels, respectively. The internal solution contained 2 mm ATP to prevent run-down of the channels (Fakler, riindle, Glowatzki, Zenner & Ruppersberg, 1994b). Arrowheads in b show the subconducting levels. Leak and capacitive currents were eliminated by subtracting the current records where the channels had run down after exposure of the patch to the internal solution containing 5 mm Mg2+ and no ATP., currents elicited by a ramp voltage pulse from -100 to +50 mv over a duration of 1t5 s in an excised patch containing many IRK2 channels. The Mg2+ concentration of the perfused solution was progressively increased. Leak and capacitive currents were subtracted, as in A. C, fraction of blocked current at +40 mv is shown as a function of free Mg2+ concentration (n = 5). The curve superimposed on the data is the leastsquares fit to 'blocked/i4ontrol = 1/(1 + ([free Mg2+]/Kd)fH), where 'blocked represents currents blocked by Mgi, and IControl represents currents through IRK2 in the control. Fitted values for the dissociation constant (Kd) and the Hill coefficient (nh) were - 1 1,llM and - 1, respectively.
6 148 T Yamashita and others J Physiol A Control C J - -I- - -~~~~~~~~ nlm spermine.2 s 6} : nm spermine c) a: 0) nm spermine o Spermine concentration (nm) -40 mv nm spermine pa mv 100 ms Spermine concentration (nm) Control 1 nm spermine 3 nm 100 nm -40 mv +40 mv 100 pa Figure 4. The effects of internal spermine on the expressed IRK2 channel A, various concentrations of spermine (1, 3, 10 and 100 nm) were perfused to an inside-out patch containing two IRK2 channels with Mg2+-free internal solution. Voltage steps to +40 mv for 200 ms were applied from the holding potential of -40 mv. The dashed lines indicate the zero current level. The leak and capacitive currents were subtracted as in Fig. 3., the effects of internal spermine on the current through an excised patch containing many IRK2 channels. Voltage steps to +40 mv for 200 ms were applied from the holding potential of -40 mv. The current traces were averaged currents of ten records. The dashed line indicates the zero current level. While spermine < 100 nm had almost no effects on the current at -40 mv, it blocked IRK2 channels in a concentration- and time-dependent manner at +40 mv. The leak and capacitive currents were subtracted as in Fig. 3. C, upper panel, the fraction of blocked current at the end of the command pulse at +40 mv is shown as a function of the spermine concentration (n = 5). The curve superimposed on the data is the least-squares fit to Iblocked/Icontrol = 1/(1 + ([spermine]/kd)fh). Fitted value for nh was between 1-6 and 2. IC50 was -3 nm. C, lower panel, the reciprocals of the time constants of the current decay were plotted against the concentration of spermine and polynomially fitted. The order of the fitted polynomials was -1-8, which was well correlated with the Hill coefficient.
7 J Physiol Mg2+ and spermine competition for IRK2 channels 149 The left panel of Fig. 5 shows the relationship between the membrane potential and the open probability (PO) of the IRK2 current in the presence of 10 nm spermine. In this graph, the PO is expressed as a relative value normalized to the current size measured at the end of command pulses in the absence of spermine. The data were obtained from three different patches. The relationship was fitted by the oltzmann function as follows: 9K = 1/(l + exp(ze( Vm -V.5)IkT)), where Vm is the membrane potential, V0.5 is the halfblocking potential under 10 nm spermine, z is the effective valence (= charge x electrical distance). The continuous line in the graph is the fitted curve with V0.5 of 9f22 mv and z of These values of VO.5 and z are similar to those reported for IRK1 channels (Lopatin et al. 1994). The time constant of current alteration was also obtained by fitting (1) A Control 10 nm spermine 100 pa I t- L, _ Am!.o _~ ~ ~ ~~~~.d 1% ~~ms -100 mv 0 cc 1*0 _ I I I I I Voltage (mv) loor 50 1 Un E If- 0' Voltage (mv) Q Figure 5. Voltage-dependent effects of spermine on the expressed IRK2 channel A, currents elicited by a series of voltage steps in an excised patch (left panel, control; right panel, under application of 10 nm spermine). The voltage steps for 200 ms were applied in 10 mv increments from the holding potential of 0 mv (upper panel). To examine the unblocking process of spermine, the voltage steps to various potentials between +10 and -100 mv were applied just after a conditioning voltage step to +40 mv for 200 ms (lower panel). These current traces revealed the time- and voltage-dependent effects of spermine on the expressed IRK2 channel. The leak and capacitive currents were subtracted as in Fig. 3., relative open probability (PO; left) and time constant of relaxation of the current (T; right) in the presence of 10 nm spermine were plotted against membrane potentials. In the left panel, relative P. was fitted with a oltzmann function (see text for details).
8 150 T Yamash ita and others J Physiol the current with a single exponential curve at each potential. As depicted in the right panel of Fig. 5, the voltage dependency of the time constant showed a peak of the time constant around +10 mv, consistent with that of the activation gating of cardiac background inwardly rectifying K+ channel (Kurachi, 1985; Fakler et al. 1994a; Ficker et al. 1994; Lopatin et al. 1994). This observation also suggests that spermine underlies the 'intrinsic gating' of inward rectifiers. Competition between Mg"+ and spermine for blockade of outwardly flowing IRK2 current ecause both Mg2+ and spermine physiologically exist in the cytosol, it is important to examine the interaction between these two substances in blocking of the outward IRK2 currents as reported by Fakler et al. (1994a) (Fig. 6). At +40 mv, Mg2+ (20 /SM) instantaneously and spermine (10 nm) time-dependently blocked the outward IRK2 current (Fig. 6A). At the end of the command pulse, the outward current was completely blocked by spermine but A -40 mv I +40 mv Control 1m0 nm spermine -20 mv 10 nm spermine + 20 um Mg?+ -I I... N.% N.% N.% 50 pa 50 ms. l10 nm spermine 10 nm spermine + 20 /um Mg2+I 20,uM Mg?+I Control 5 ms Figure 6. Effects of Mg"+ and spermine alone and combined on the IRK2 channel in an excised patch Command pulses to +40 mv for 200 ms and thereafter to 20 mv for 200 ms were applied from the holding potential of -40 mv. The concentrations of free Mg2+ and spermine were fixed at 20 /M and 10 nm, respectively. The internal solution contained 2 mm ATP to prevent channel run-down. The zero current level is indicated by the arrow. Data were obtained at a filter of 3 khz and a sampling rate of 10 khz. The leak and capacitive currents were subtracted as in Fig. 3. A, perfusion with 20 um free Mg2+ alone caused a fast block followed by a minimal slow block of the outward currents through IRK2 channels. Spermine alone blocked outward current in a time-dependent manner. However, adding Mg2+ to spermine slowed the current decay, and subsequently increased the current at the end of the depolarizing pulse, compared with that under application of spermine alone., upon hyperpolarizing pulses to -20 mv, an instantaneous inward current was followed by a slowly activating one (time constant, -I ms) under application of 20 #M Mg2' alone. The former and the latter would represent fast and slow unblocking processes, respectively. In contrast, under 10 nm spermine alone, the hyperpolarizing pulse activated an inward current (time constant, -0 4 ms) without significant instantaneous components. Under application of their combination, the pulse elicited an instantaneous inward current followed by an activating one which was fitted by the sum of two exponentials (time constants, -0 4 and -1 ms, respectively).
9 J Physiol Mg2+ and spermine competition for IRK2 channels 151 not by Mg2+. When both Mg2+ and spermine were applied to the patch membrane, the virtually instantaneous outward current evoked by a depolarizing pulse to +40 mv was the same level as that in the presence of Mg2+ alone, but the current gradually decreased to an intermediate level between those of Mg2+- and spermine-blocked currents during the command pulse, indicating that Mg2+ and spermine interact with each other on the outwardly flowing IRK2 current. The effects of combination of Mg2+ and spermine on unblocking process was also examined with the hyperpolarizing pulse to -20 mv (Fig. 6). In the presence of Mgi+ alone, the hyperpolarizing pulse resulted in an almost instantaneous unblocking which was followed by a small time-dependent increase in the inward current (time constant, 1 ins). This slow Mg2+ unblock was similar to that reported in cardiac background inwardly rectifying K+ channel and cloned IRK1 channels (Matsuda, 1988; Fakler et al. 1994a). On the other hand, a large timedependent increase in the inward current (time constant, -0 4 ms) was observed in the presence of spermine alone. When both substances were present, the size of the instantaneous inward current was between the former two values. The time-dependent component could be fitted by the sum of two exponentials with the time constants of 0 4 and 1 ms. Although the time constants were quite small for appropriate evaluation with curve fitting and therefore may include some errors, these values of time constants were the same as those estimated individually for the unblocking processes of Mg2+ and spermine, respectively. These results suggest, therefore, that Mg2+ and spermine may bind to the channel independently. We examined the voltage dependence of the interaction of Mg2+ and spermine on the IRK2 channel. Figure 7 shows sets of current traces evoked by voltage steps to various potentials between -100 and +40 mv in the presence of Mg2+ and/or spermine. The patch contained more than ten channels. The decay of the outwardly flowing IRK2 currents in the presence of spermine and Mg2+ was slower at +30 and +40 mv than in the presence of spermine alone (time constant at +30 mv: vs. 68 ± 11 ms, P< 0 05; at +40 mv: vs ms, P< 0-01, n = 4. See also Fig. 8). Outwardly flowing IRK2 currents at the end of the command pulses were larger than those in the presence of spermine alone at potentials of +30 and +40 mv (Fig. 7; +30 mv: vs pa, A Control 20.M Mg?+ 10 nm spermine 20,uM Mg? nm spermine -=s- 0- C I Voltage (mv) 50 mqs J 100 pa Figure 7. Voltage dependence of the effects of Mg"+ and spermine alone and combined on the expressed IRK2 channel A, examples of current traces evoked by a series of voltage steps. The command pulses for 200 ms were applied in 10 mv increments between -100 and +40 mv from the holding potential of 0 mv. The leak and capacitive currents were subtracted as in Fig. 3., current-voltage relationships at the end of the command pulses under control (Mg2+ free, 0), 20 /M free Mg2+ (A), 10 nm spermine (El) and 20 um free Mg2+ and 10 nm spermine combined (0). Currents at potentials between -60 and +40 mv are shown. Those at more negative potentials showed no significant change from the control value.
10 152 1T Yamashita and others J Physiol mV: vs. 11+lOpA, n=4, P<005, respectively). Thus, Mg2+ attenuated the negative slope of conductance created by spermine at depolarized potentials. Neither Mg2+, spermine, nor their combination induced any significant changes in the steady-state inward currents. The unblocking process of the Mg2+- and spermine-induced blocks at hyperpolarized potentials shown in Fig. 6 suggests a competitive interaction between these two substances on the outwardly flowing IRK2 currents. Therefore, we analysed the interaction of these substances using the following simple model for the description: 4[Mg2+] 0z'[Sp]nH Mg - /3/' Model 1. 0 K sp, where 0 represents the unbound state of the channel; Mg, Mg2+-bound state; 5p, spermine-bound state; a and a', association rate constants; /1 and /', dissociation rate constants; nh, Hill coefficient for spermine; [Mg2+], concentration of Mg2+; [Sp], concentration of spermine. With regard to Mg2+ block, we dealt with only Mg2+induced fast block for simplicity, because the slow block A 2.5r Mg" Spermine Co *0 F < 200 Cl) ui U) -I Voltage (mv) Voltage (mv) 20,oM Mg nm spermine +40 mv mv t;, +20 mv mv _ 4% 20 pa Command voltage (mv) (ms) Predicted 7r (ms) e e? C) (D ms Voltage (mv) Figure 8. Model of the effects of Mg"+ and spermine combined on IRK2 channels A, association and dissociation rate constants for Mg2+-induced fast block (a and /l, respectively, left panel) and spermine-induced block (a' and,', respectively, right panel) of the IRK2 channel. The mean values of three different patches are plotted. Rate constants were computed according to the method shown in the text. locked fractions were estimated from the rapidly decaying component of the outward current for Mg2' and the current at the end of the depolarizing command pulses for spermine. The time constants were estimated by fitting the current decay to a single exponential function. These data were used for calculating the rate constants (see text), which were plotted against membrane voltage. In our experiments EK was 0 mv, therefore, Vm should represent the voltage shift from EK. The rate constants could be fitted to single exponential functions: Mg2+: 0, 3-2 x 106exp(0.049 Vm); Q, 5.4 x 102exp( Vm); spermine: 0, 1P2 x 1015exp(0 030 Vm); 0, 5-1 x 10exp(-0.11 Vm)., left panel, experimental current traces (points) and simulated current decays (continuous curves) at potentials of +10, +20, +30 and +40 mv under application of 20 #M free Mg2+ and 10 nm spermine in an excised patch containing many IRK2 channels. Although the relaxation should be a bi-exponential function by the model, the computed fast time constants were too rapid (< 0 3 ms) to be determined from the experimental current traces. Middle panel, comparison between experimental and predicted time constants at +10, +20, +30 and +40 mv. The concentrations for Mg2+ and spermine were at 20 /M and 10 nm, respectively. Right panel, the predicted current-voltage relationships of IRK2 channels in the presence of 20 um Mg2+ (A) and/or 10 nm spermine (o) are depicted; 0, control; 0, 20 /M Mg2+ and 10 nm spermine combined. These relationships are almost identical to those obtained experimentally (see Fig. 8). The predicted relative current under 20 FM Mg2+ was somewhat larger than the experimental one, which is probably because the Mg2i+-induced slow block was disregarded in the model.
11 J Physiol Mg2+ and spermine competition for IRK2 channels 153 induced by [Mg2+]1 of > 10 /M was minimal as shown in Figs 6 and 7. The voltage-dependent rate constants for the two substances were calculated from the block of the IRK2 channel by Mg2+ or spermine alone as follows. 1/T = a x cm+ (2) blocked fraction = a x cm/(a x ctm + /3), (3) where m,, and c represent the Hill coefficient, time constant of the current decay and concentration of the ligand (Mg2+ or spermine), respectively. From the above equations, the rate constants at several different concentrations of Mg2+ (10, 20, and 30 /SM) and spermine (1, 3, 10, 30 and 100 nm) were calculated at potentials between +10 and +40 mv. The Hill coefficient was fixed at 1 for Mg2+ and 1P8 for spermine. Time constants for Mg2+ block were estimated by fitting single exponential curves with the data filtered at 3 khz (-3 d) and sampled at 10 khz. The time constants might contain some errors originating from too rapid time course of the current, but was considered as a reasonable estimate of Mg2+ block for the simple model. According to the data from three experiments, the rate constants at each of the potentials did not significantly vary even if the different concentrations of these substances were used (Fig. 8A). For spermine, rate constants could also be estimated by a fitting of 1/r versus spermine concentration with a polynomial function (eqn (2) only). The estimated values were almost the same as those obtained by eqns (2) and (3). From these results, it is likely that the steep voltage dependence of spermine block mainly results from the substantial voltage-dependent change in its dissociation rate constant. In model 1, the channel is assumed to acquire three possible states in the presence of Mg2+ and spermine. Therefore, it is predicted that the relaxation of the outward IRK2 current should comprise two exponentially decaying currents. The time constants of these two exponentials should be obtained as the solutions of the following quadratic equation, while the amplitude of each component could be calculated as described by Colquhoun & Hawkes (1977). A2 +pa+ q=0, (4) -p = a[mg21] +, + a' [Sp]l8 +,', (5) q = a[mg]2 ' + /3/ + X [Sp] l8/3 (6) where A is the reciprocal of the time constants of the two exponentials. The solutions of the quadratic equation, Al and A2, should be the reciprocals of the two time constants. From these equations, the time constants in the presence of 20 /tm Mg2+ and 10 nm spermine were calculated at +10, +20, +30 and +40 mv. The current relaxation at each potential was predicted with these time constants (Fig. 8, left panel). The predicted relaxation fitted the real current trace well at each potential. The table in the middle panel of Fig. 8 compares the time constants for the slow component predicted by the model with those estimated from the fitting of the currents. Although the relaxation based on this model should be fitted by double exponentials, the time constant of the fast component was too small (< 0 3 ms) to allow any reliable fitting. In the table, therefore, the comparison is made only for the slow component. The two values are in good agreement at each potential. The current-voltage relationships at the end of the 200 ms command pulses in the absence and the presence of Mg2+ and spermine were also predicted from the model (Fig. 8, right panel). The negative slope conductance region was formed at potentials between +20 and +40 mv in the presence of spermine alone and also in the presence of both Mg2+ and spermine. The slope conductance was less negative in the presence of both substances than in the presence of spermine alone. The region was not evident in the presence of Mg2+ alone. Thus, Mg2+ apparently attenuated the inward rectification evoked by spermine. The predicted current-voltage relationships under various conditions simulate well those obtained from the experimental current recordings (Fig. 8). The model also predicts that, at 1 ms after the onset of depolarizing pulse, the current amplitude in the presence of both spermine and Mg2.+ is almost identical to that in the presence of Mg2+ alone, while at the steady state the current level will become identical to that in the presence of spermine alone. Therefore, the observed deceleration of the spermineinduced relaxation by Mg2+ may result from a timedependent replacement of Mg2+ with spermine on the channel molecule. DISCUSSION The major findings in the present study are as follows. (1) An inwardly rectifying K+ channel encoded by the IRK2 clone can be efficiently expressed in a human cell line (HEK 293T). Its electrophysiological properties were similar to those expressed in Xenopus oocytes (Takahashi et al. 1994). (2) oth Mg2+ and spermine applied to the intracellular side of the patch membranes blocked the outwardly flowing IRK2 channel currents in a concentrationdependent manner. However, the effects of these two substances differed in their time and voltage dependencies, positive co-operative interaction with the channel and induction of subconducting levels of the channel current. (3) Mg2.+ apparently attenuated the inhibitory effects of spermine on the channel. The interaction between Mg2+ and spermine could be interpreted in terms of the mutually exclusive binding of the two substances to the channel. lock of the outward current through IRK2 channels by Mg2+ and spermine Mg2+ has been reported to cause the inward rectification of cardiac background inwardly rectifying K+, KACh and
12 154 T Yamashita and others J Physiol KATP channels besides intrinsic gating of the channels (Horie et al. 1987; Horie & Irisawa, 1987; Matsuda et al. 1987; Vandenberg, 1987; Matsuda, 1988). Recently, a cloned inward rectifier channel, IRK1, has been reported to be blocked by Mg2+ in a voltage-dependent manner (Stanfield et al. 1994a; Taglialatela et at. 1994). Since the IRK2 channel is expressed in the heart and its deduced amino acid sequence shows high homology (70%) with that of the IRK1 channel (Takahashi et al. 1994), block of IRK2 channels by Mg2+ shown in the present study is compatible with the observation that cardiac background inwardly rectifying K+ channel is blocked by Mg2+ (Matsuda et al. 1987; Vandenberg, 1987). In IRK2 channels, Mg2+ evoked subconducting levels of outwardly flowing channel currents at depolarized potentials. oth fast and slow blocks were observed in the presence of low concentrations of Mg2+, but the fast block became predominant at higher concentrations. No positive co-operativities were observed in the concentration-response relationship of the Mg2+ block. These observations are the same as those reported on the cardiac background inwardly rectifying K+ or IRKI channel (Matsuda, 1988; Taglialatela et al. 1994). Spermine physiologically exists in the cytosol of various cells and was recently reported to cause inward rectification of IRKI and cardiac KACh channels by acting as a gating substance (Fakler et al. 1994a; Ficker et al. 1994; Lopatin et al. 1994; Yamada & Kurachi, 1995). Spermine also gated the IRK2 channel in a voltage- and time-dependent manner. The potency of spermine in gating of IRK2 channels (IC50, -3 nm at 40 mv) was similar to that in IRK1 channels (IC50, -8 nm at 40 mv). Spermine, however, inhibited IRK2 channels in a positive co-operative manner, which was not detected in IRK1 channels (Ficker et al. 1994). This difference might result from the differences in the structure between IRK1 and IRK2 channels or in the utilized expression systems. Competition between Mg2+ and spermine Although both Mg2+ and spermine blocked the outwardly flowing IRK2 currents, these two substances acted on the channel in different ways. Spermine, in comparison with 2+ exhibited slower time-dependent and steeper voltage-dependent block. Kinetical analysis of the relaxation of IRK2 current induced by these substances revealed that the difference in the voltage dependence of the dissociation rate constants is mainly responsible for these differences. From these observations, it is suggested that the spermine-bound state of the IRK2 channel is different from the Mg2i+-bound state. Since both Mg2+ and spermine physiologically exist in the cytosol, these substances would interact with the channel concomitantly. Model 1, which assumes a competitive interaction of these substances on the channel, described well the actual current relaxation. Therefore, it could be concluded that spermine and Mg2+ may share the same binding site(s) on the channel i (Fakler et al. 1994a; Ficker et al. 1994; Lopatin et al. 1994; Stanfield et al b). According to the present data, Mg2.+ blocks the channel with much faster kinetics than spermine. Thus, on abrupt depolarization of the membrane, the binding site of the channel would be first occupied by Mg2+ and then by spermine. Similar competitive binding is demonstrated in cardiac Na+ channels under application of two different antiarrhythmic drugs (Clarkson & Hondeghem, 1985; Kohlhardt & Stefert, 1985). Lidocaine is reported to attenuate blockade of the Na+ channel by bupivacaine, which has slower kinetics than lidocaine. The effect of the drug combination is mainly attributed to the differences in their offset kinetics. In the present results, however, the difference in the onset kinetics between Mg2+ and spermine appeared to play a major role. Mg2+ with the faster onset kinetics apparently attenuated the effects of spermine which has slower onset kinetics, consistent with the previous observation that the relaxation of the cardiac background inwardly rectifying K+ channel evoked by the 'intrinsic gating' is slowed by Mgi+ (Ishihara et al. 1989). The relaxation observed in the presence of both Mg2+ and spermine was nicely predicted by the model, which assumes that the two substances competitively bind to the channel with independent kinetics. ased on this model, the current relaxation may be explained as follows. Upon depolarization, Mg2+ instantaneously blocks some fraction of the channels, which reduces the number of the channels available for spermine. However, spermine subsequently replaces Mg2+ on the channels, and the equilibrium is accomplished between the channel, Mg2+ and spermine according to the rate constants of individual reactions determined by membrane potentials. This model offers a simple description and, therefore, may be oversimplified in some areas. In fact, Mg2+ obviously interacts with the IRK2 channel in a more complex way, as shown by its fast and slow blocking processes. Spermine has also been reported to have some time-independent effects on a mutant channel of IRK1 (Ficker et al. 1994). Despite these ambiguities, our present model could satisfactorily predict the current-relaxation and the current-voltage relationships. Therefore, it is plausible that the inward rectification of the IRK2 channel results mainly from the block of the channel by Mg2+ and spermine, both of which bind to the channel in an independent and mutually exclusive manner. It was recently proposed that a single negatively charged residue, i.e. an aspartate at position 172 in the putative second transmembrane segment (M2) of the IRKI channel, plays an important role in the interaction of both Mg2+ and spermine with the channels (Fakler et al. 1994a; Ficker et al. 1994; Lopatin et al. 1994; Stanfield et al. 1994b). It is also known that the IRKI channel has another binding site for Mg2+ in its carboxyl terminus (Taglialatela et al. 1994;
13 J: Physiol Mg2+ and spermine competition for IRK2 channels 155 Wible, Taglialatela, Ficker & rown, 1994). Since the IRK2 channel also has an aspartate residue at the corresponding site in its M2 region, the site may be a candidate for the interaction between Mg2+ and spermine described in this study. The high amino acid sequence similarity between IRK1 and IRK2 channels suggests that the carboxyl terminus of IRK2 may also interact with Mg2+. Therefore, the further improvement of the present model would be possible based on the experiments with, for instance, site directed mutagenesis of the IRK2 channel. Limitations Although spermine is important in the genesis of the negative slope conductance of inward rectifiers, the competition with Mg2+ would play a role in preventing the spermine-induced inward rectification from being too strong, as shown in the present study. The interaction between Mg2+ and spermine might, therefore, underlie the inward rectification in physiological conditions. However, the outward currents through the background inwardly rectifying K+ channel are probably regulated by more complicated mechanisms than described in the present study. First, concentrations of Mg2+ and spermine are both much higher in physiological conditions than those used in the present study, although the effects of higher doses are difficult to analyse for technical reasons. Second, other intracellular polyamines would contribute to the inward rectification. Spermidine, a weaker intracellular gating substance, causes a time-dependent block of the outward currents of IRK1 (Ficker et al. 1994; Lopatin et al. 1994) and KACh channel (Yamada & Kurachi, 1995). Putrescine is also known to have a time-independent blocking action on the IRK1 channel (Ficker et at. 1994). Moreover, the physiologically observed decay of the outward currents is slower than predicted by our model in the presence of physiological concentrations (> 100 /um) of polyamines. Third, the negative slope conductance of the inwardly rectifying channels varies somewhat among a variety of animal species (Leech & Stanfield, 1981; Josephson & rown, 1986; Giles & Imaizumi, 1988; Matsuda, 1988; Ishihara et at. 1989). Some works have reported that, upon depolarization to the potentials more positive than EK +30 mv, the inwardly rectifying K+ channel current was deactivated in a time-dependent manner leaving negligible currents in the steady state (Leech & Stanfield, 1981; Josephson & rown, 1986). However, others have reported the existence of maintained and non-declining prominent outward currents even at EK values between +40 and +60 mv (Giles & Imaizumi, 1988; Matsuda, 1988; Ishihara et al. 1989). These variations might arise from species differences in the structure of inwardly rectifying K+ channels (Trautwein & McDonald, 1978), and in concentrations of the cytosolic free Mg2+ and polyamines. However, these variations also raise a possibility that other unknown mechanisms might modify the competition between Mg2+ and polyamine. CLARKSON, C. W. & HONDEGHEM, L. M. (1985). Evidence for a specific receptor site for lidocaine, quinidine, and bupivacaine associated with cardiac sodium channels in guinea pig ventricular myocardium. Circulation Research 56, COLQUHOUN, D. & HAWKES, A. G. (1977). Relaxation and fluctuations of membrane currents that flow through drug operated channels. Proceedings of the Royal Society 199, FAKLER,., RXNDLE, U., GLOWATZKI, E., WELDEMANN, S., ZENNER, H. P. & RUPPERSERG, J. P. (1994a). Strong voltage dependent inward rectification of inward rectifier K+ channels is caused by intracellular spermine. Cell 80, FAKLER,., RXNDLE, U., GLOWATZKI, E., ZENNER, H. P. & RuPPERSERG, J. P. (1994b). Kir2.1 inward rectifier K+ channels are regulated independently by protein kinases and ATP hydrolysis. Neuron 13, FICKER, E., TAGLIALATELA, M., WILE,. A., HENLEY, C. M. & ROWN, A. M. (1994). Spermine and spermidine as gating molecules for inward rectifier K+ channels. Science 266, FLETCHER, G. H. & CHIAPPINELLI, V. A. (1992). An inward rectifier is present in presynaptic nerve terminals in the chick ciliary ganglion. rain Research 575, GILES, W. R. & IMAIZUMI, Y. (1988). Comparison of potassium currents in rabbit atrial and ventricular cells. Journal of Physiology 405, HAGIWARA, S., MIYAZAKI, S. & ROSENTHAL, N. P. (1976). Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish. Journal of General Physiology 67, HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN,. & SIGWORTH, F. J. (1981). Improved patch clamp technique for high resolution current recordings from cells and cell free membrane patches. Pfluigers Archiv 391, HORIE, M. & IRISAWA, H. (1987). Rectification of muscarinic K+ current by magnesium ion in guinea pig atrial cells. American Journal of Physiology 253, H HORIE, M., IRISAWA, H. & NOMA, A. (1987). Voltage-dependent magnesium block of adenosine triphosphate sensitive potassium channel in guinea-pig ventricular cells. Journal of Physiology 387, ISHIHARA, K., MITSUIYE, T., NOMA, A. & TAKANO, M. (1989). The Mg2+ block and intrinsic gating underlying inward rectification of the K+ current in guinea-pig cardiac myocytes. Journal of Physiology 419, ISHIKAWA, T., WEGMAN, E. A. & COOK, D. I. (1993). An inwardly rectifying potassium channel in the basolateral membrane of sheep parotid secretary cells. Journal of Membrane iology 131, JAN, L. Y. & JAN, Y. N. (1994). Potassium channels and their evolving gates. Nature 371, JOSEPHSON, I. R. & ROWN, A. M. (1986). Inwardly rectifying single channel and whole cell K+ currents in rat ventricular myocytes. Journal of Membrane iology 94, KOHLHARDT, M. & STEFERT, C. (1985). Properties of Vmax block of INa mediated action potentials during combined application of antiarrhythmic drugs in cardiac muscle. Naunyn-Schmiedeberg's Archives of Pharmacology 330, KOYAMA, H., MORISHIGE, K., TAKAHASHI, N., ZANELLI, J. S., FAss, D. N. & KURACHI, Y. (1994). Molecular cloning, functional expression and localization of a novel inward rectifier potassium channel in the rat brain. FES Letters 341,
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