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Am J Physiol Heart Circ Physiol 283: H2119-H2129, 2002. First published July 26, 2002; doi:10.1152/ajpheart.00326.2002
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Vol. 283, Issue 5, H2119-H2129, November 2002

G protein modulates thyroid hormone-induced Na+ channel activation in ventricular myocytes

Luyi Sen, Yoshihide Sakaguchi, and Guanggen Cui

Division of Cardiology, Department of Medicine, The David Geffen School of Medicine, University of California, Los Angeles, California 90095


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To evaluate the effects of liothyronine (3,5,3'-triiodo-L-thyronine, T3) on Na+ channel current (INa) properties, INa was recorded in adult guinea pig ventricular myocytes. T3 (1 nM) acutely increased whole cell INa and shifted the steady-state INa inactivation curve dose dependently. When the pipette solution contained 100 µM GTP or GTPgamma S, the effect of T3 on the whole cell INa was increased two- to threefold. This effect was almost completely abolished by pertussis toxin preincubation. In the cell-attached patch, T3 increased the open probability of single INa by reducing the null probability. In the inside-out patch, T3 effect was 10 times faster than that in whole cell and cell-attached patches while GTPgamma S was present and could be completely washed out. T3 alone slightly increased the channel open probability by increasing the closed state to open state rate constant (kCO) and reducing the null probability. GTPgamma S exposure only increased the number of functional channels. T3 and GTPgamma S synergistically enhanced the channel open probability 5.8 ± 0.5-fold by increasing kCO, decreasing the open state to absorbing inactivated state rate constant, and greatly reducing the null probability. These results demonstrate that T3 acts on the cytosolic side of the membrane and acutely activates INa. Pertussis toxin-sensitive G protein modulation greatly magnifies the T3 effects on the channel kinetics and null probability, thereby increasing the channel open probability.

T3; pertussis toxin-sensitive G protein; whole cell sodium channel current; single channel current; cardiac myocytes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THYROID HORMONES play an important role on cardiac electrophysiological properties (3, 6, 42). The effects of thyroid hormones have been considered to exert their actions through nuclear and/or extranuclear mechanisms. Nuclear effects are mediated by the binding of thyroid hormones to specific nuclear receptors, resulting in increased transcription of thyroid hormone-responsive cardiac genes (16, 34). Extranuclear effects, which are independent of nuclear effects, primarily influence the transport of amino acids and sugars through the cell membrane (5, 39). Recently, specific binding sites for thyroid hormones on the plasma membrane have been reported (15, 40). However, few studies (4, 7, 43) have demonstrated the effects of thyroid hormones on cardiac ionic channels. We (37) have previously reported that liothyronine (3,3',5-triiodo-L-thyronine, T3) enhanced single inward rectifier K+ channel currents by increasing the channel open probability (Po) in cell-attached patches. Rubinstein and Binah (36) reported that Ca2+ channel currents and delayed rectifier K+ channel currents are increased in hyperthyroidism, and Ca2+ channel currents are decreased in hypothyroidism in guinea pig myocytes. Subsequently, T3-induced increase in cardiac L-type Ca2+ current was observed in in vitro studies (20, 33). It has also been found that acutely administered T3 promoted slow inactivation kinetics of the Na+ channel current (INa) in neonatal rat myocytes (19). Dudley et al. (7) reported that T3 quickly induced INa bursting in cell-attached patches in rabbit ventricular myocytes. However, the molecular mechanism of the effect of thyroid hormone on the ion channels in cardiac myocytes is not well understood. In this study, we explored the detailed mechanisms of G protein-modulated acute T3 action on INa in ventricular myocytes.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation. Single ventricular myocytes were isolated from adult male guinea pig (250-350 g) hearts using a modified procedure, as previously described (34). In brief, the guinea pigs were anesthetized with ether. Hearts were rapidly excised, mounted on a Langendorff-type apparatus, and perfused retrogradely through the aorta at 37°C. Hearts were perfused for 5 min with nominally Ca2+-free Tyrode solution containing (in mM) 136 NaCl, 5.4 KCl, 0.3 NaH2PO4, 1.0 MgCl2, 10 HEPES, 4D-mannitol, 0.6 thiamine HCl, and 5.5 glucose (pH 7.4 with NaOH). Hearts were then perfused for 4-6 min with Ca2+-free solution containing 1 mg/ml type I collagenase (Sigma; St. Louis, MO), 1 mg/ml bovine albumin (Sigma), and protease (7.6 mg/50 ml, Sigma). Thereafter, the collagenase was washed out with a high-K+ and low-Cl- solution, which was composed of (in mM) 115.9 KOH, 80 glutamic acid, 10 taurine, 14 oxalic acid, 10 KH2PO4, 10 HEPES, 25 KCl, 0.5 EGTA, and 11 glucose (pH 7.4 with KOH). The perfusion rate was ~5-8 ml/min. Ventricular cells were gently dispersed and immersed in 0.1 mM Ca2+-Tyrode solution with albumin (1 mg/ml, bovine, Sigma), which was made by the addition of 0.1 mM CaCl2 to Ca2+-free Tyrode solution. After 10-20 min, 1.7 mM CaCl2 was added to the above solution, and cells were stored in this solution (1.8 mM Ca2+). All solutions were aerated with 95% O2-5% CO2. Only the cells that were Ca2+ tolerant, rod shaped, and having clear striations without any blebs on the surface were selected for the experiments.

Whole cell patch-clamp recording. Na+ current was recorded from single cardiac myocytes by the whole cell patch- clamp technique described by Hamill et al. (18). An axopatch-1D amplifier (Axon Instruments; Foster City, CA) was used for the voltage-clamp experiments. Protocol generation and data acquisition were controlled with an IBM computer and 12-bit analog-to-digital and digital-to-analog converter by using pCLAMP software. The internal solution contained (in mM) 130 CsCl, 10 NaCl, 2 Mg2Cl, 5 Cs2-EGTA, 10 HEPES, and 4 ATP (pH 7.4). The external solution contained (in mM) 40 NaCl, 95 tetraethylammonium (TEA), 5.4 CsCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4).

Single channel recording. Single INa in ventricular myocytes were recorded by using cell-attached patches, according to the patch-clamp method described by Hamill et al. (18). Pipettes were fabricated from thin-wall glass and were coated near the tip with silicon (Sylgard, Dow Corning) to reduce the capacitance to ground and were then heat polished. When the pipettes were filled with the solution, the resistance of pipettes was 8-15 MOmega . The sealing resistance after negative pressure was applied to the inside of the pipette (~30 cmH2O) was >30 GOmega . Signals for single channel currents were recorded with the patch-clamp amplifier (Axopatch-200B, Axon Instruments) and stored on a hard drive of an IBM computer and optic disk. Currents were sampled every 50 µs and recorded signals were filtered with a cutoff frequency of 2 kHz (-3 dB, four-pole low-pass Bessel filter). Patches were held at -140 mV and depolarized once per second to test potentials (VT) (-60, -50, -40, and -30 mV) for 50 ms. At least 300 sweeps (5 min) were recorded at each VT. Patches with four channels or less were used for this study. Patches that showed obvious rundown were omitted. Control data were recorded at least 5 min after the gigaohm seal was obtained, as time-dependent changes in kinetics of single INa were remarkable during the first 5 min after seal formation. To estimate time-dependent changes in kinetics of single INa, cells were perfused with an external solution without T3 and data were recorded 0-5, 5-10, 15-20, and 25-30 min, respectively, after the formation of gigaohm seals at a VT of -40 mV. Data that could be recorded for 20 min after the formation of gigaohm seals without deterioration or loss of gigaohm seals were used for analysis. All recordings were obtained at room temperature (22-24°C). For cell-attached recordings, isolated cells were superfused with a high-K+ external solution of the following composition (in mM): 115.9 KOH, 80 glutamic acid, 14 oxalic acid, 10 KH2PO4, 10 HEPES, 25 KCl, 0.5 EGTA, and 11 glucose (pH 7.4 with KOH). The resting potential for these cells was assumed to be 0 mV and no correction was applied for the holding potential and VT. The high-K+ external solution with 1 µM T3 was adjusted pH (7.4) after the addition of T3 with KOH. Pipette solution contained (in mM) 140 NaCl, 1 CaCl2, 5 MgCl2, 10 TEA chloride hydrate, and 10 HEPES (pH 7.4 with NaOH). Single channel inside-out patch bath solutions contained (in mM) 140 K+ aspartate, 1.0 EGTA, 0.55 CaCl2, 2.0 Mg2+ ATP, and 10 HEPES (pH 7.25).

Experimental protocols and data analysis. The voltage dependence of the steady-state inactivation relationship was examined with a standard two-pulse protocol. A VT of -20 mV was preceded by a 500-ms preconditioning pulse ranging from -150 to +20 mV with a 0.1-Hz pulse interval. Under these conditions, the slow inactivation-dependent shift in the inactivation curves was minimized. The normalized curves were fitted using a Boltzmann distribution equation.

Single channel currents from patch-clamp recordings were analyzed using the software program pCLAMP (version 6.01, Axon Instruments). Recordings were corrected off-line for leak and capacitive currents by analog circuitry and by subtracting the average of recordings without channel openings (null sweeps) at each given potential. Openings detected by the computer were confirmed by visual observation and used for further analysis. The threshold to judge the open state was set at half of the unit amplitude of the single channel current. The number of channels in each patch was estimated from channel overlap at the test potentials of -20 to -10 mV over 1,000 sweeps. Open time histograms were constructed from the data in which opening events with overlapping openings of two or more channels were excluded. The first bin was omitted to fit open time histograms because dead time for data filtered at 2 kHz was 90 µs. Ensemble currents were calculated by the following equation: 1,000 (sum of sweeps)/[(no. of channels)(no. of sweeps)] as the currents of 1,000 channels. Null probability (PN) was calculated by taking the nth root of the fraction of observed null sweeps (where n represents the estimated number of channels in the patch). Po plots were constructed by averaging the probability of openings at each time per one channel with the number of sweeps. Assuming that each channel opening shows independent identical distribution, the number of openings per one sweep with openings was calculated by the following equation: (averaged no. of openings per one sweep)/[(1 - PN)(no. of channels)].

Statistics. All data were expressed as means ± SD unless otherwise indicated. Statistical significance was evaluated by Student's paired and unpaired t-test, where appropriate. Differences with a value of P < 0.05 were considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

T3 increases whole cell INa. T3 (1 nM) significantly increased whole cell Na+ current density by 10 ± 2% compared with that in control conditions at a holding potential of -100 mV (n = 18, P < 0.05). Figure 1A illustrates the acute effect of 10 nM T3 on the whole cell INa. At a VT of -10 mV and a holding potential of -100 mV at 24°C, the peak INa increased 47%. As shown in Fig. 1B, T3 increased the peak of the current-voltage (I-V) relationship curve, but did not change the shape of the curve or the reversal potential. After exposure to 10 nM T3, the steady-state availability curve for whole cell INa was slightly but significantly shifted to the right (3.4 ± 0.9 mV, n = 18, P < 0.05, Fig. 1C). As shown in Fig. 1D, the effect of T3 on whole cell INa was dose dependent. The EC50 of T3 was 8.7 nM. The activation curve was slightly shifted to the left by T3 (0.9 ± 0.4 mV), but this change was not statistically significant (n = 18, P = 0.058, data not shown).


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  		Fig. 1.   A: 3,5,3'-Triiodo-L-thyronine (T3; 10-9 M) induced significant increases in whole cell Na+ current (INa). A family of INa traces was elicited by a 30-ms depolarizing clamp step from a holding potential of -100 mV at test potentials (Vm) from -90 mV to +40 mV. B: current-voltage relation of whole cell INa at steady state before and after T3. open circle , Data recorded in control; , data recorded 25 min after superfusion with T3 (n = 18). C: steady-state availability curve for INa under control conditions and after exposure to 10-8 M T3. The currents were elicited by a test pulse of 20-ms duration to -20 mV from various holding potentials (Vm). D: dose-response relationship between whole cell INa and T3. I/Icon, peak current/peak current at the control condition.

Reverse 3',5',3-L-triiodothyronine (rT3) (10 nM), which has been reported to partially compete with T3 and tetraiodothyronine on the membrane receptors, did not increase whole cell INa (Fig. 2A). In cells preincubated with 10 nM rT3 (30 min), the T3 (10 nM) effect on the whole cell INa at -40 mV was blocked by 79% (P < 0.05) 25 min after superfusion with T3 was started. If cells preincubated with rT3 were then washed for 2 h, the effect of T3 could be partially restored (37%) (n = 8, P < 0.01).


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  		Fig. 2.   A: modulation of whole cell INa by T3, reverse 3',5',3-L-triiodothyronine (rT3), or 3,5,3'-L-triiodothyroacetic acid (TAA). The maximum T3 effect on whole cell INa was recorded 25 min after the cells were superfused with 10 nM T3. The percentage of increase in INa was compared with the recordings made before the T3 exposure in the same patches. B: time course of whole cell INa density enhancement by 10 nM T3 with or without GTP (100 µM) was added into the pipette solution. The arrow indicates the start of T3 superfusion.

3,5,3'-Triiodothyroacetic acid (TAA) (10 nM), an analog of T3, significantly increased whole cell INa currents in a manner similar to T3 (Fig. 2A) (37). The increment in INa caused by TAA was the same as the increment caused by a comparable concentration of T3 (62% ± 8% at -40 mV and 45 ± 7% at VT = -100 mV, n = 5).

G protein modulates effect of T3 on whole cell INa. As shown in Fig. 2B, the effect of T3 could be observed 8 min after cells were exposed to 10 nM T3 and reached a steady state in ~25-35 min, but these effects could not be washed out during the subsequent 30 min of observation. Therefore, the detailed time-dependent change using a control solution without T3 was evaluated. There was no significant change in whole cell INa current observed during 35 min of superfusion, but significant time-dependent rundown was observed at 45 min after superfusion was started (data not shown). These tendencies were observed at either temperature (24 and 37°C). As shown in Fig. 2B, when the pipette solution contained 100 µM GTP, the onset of T3 (10-8 M) effect on INa was significantly earlier. The time to one-half peak response was 12.7 ± 2.2 min with GTP compared with 21.8 ± 3.7 min without GTP (n = 15, P < 0.01). The maximum effect was reached within 17-21 min after T3 exposure.

Figure 3A shows the current-voltage relation for the tetrodotoxin-sensitive INa effected by T3 recorded with either 100 µM GTP or 100 µM GTPgamma S in the pipette. When GTP or GTPgamma S was present in the pipette solution, the peak I-V curves were increased 73 ± 11% or 75 ± 14% by T3 compared with control conditions. These increments were twofold greater than that recorded in the normal pipette solution. Our preliminary study has shown that incubation of myocytes with 200 ng/ml pertussis toxin at 33°C for 3 h resulted in virtually complete ADP ribosylation of sensitive G protein by endogenous NAD+ (41). Therefore, the same amount of pertussis toxin was used for preincubating myocytes. Pertussis toxin abolished most of the effect of T3 on the INa when either GTP or GTPgamma S was present in the pipette solution. The voltage dependence of the T3 (10 µM) effect on INa inactivation was recorded when the pipette solution with or without GTP or GTP-gamma S. When GTP or GTP-gamma S was present, T3 significantly shifted the inactivation curve toward a more positive potential (7 ± 2 mV, n = 15, P < 0.05). This effect was significantly greater than that seen without GTP or GTP-gamma S (P < 0.05) (Fig. 1C).


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  		Fig. 3.   A: peak current-voltage (V) relation for INa in control cells. (), cells 25 min after 10 nM T3 exposure without (open circle ) or with 100 µM GTP (down-triangle) or GTPgamma S () added to the pipette solution, and cells pretreated with pertussis toxin (PTX) for 2 h and then exposed to 10 nM T3 with 100 µM GTP contained in the pipette solution (triangle ). Each point is the mean ± SD of 15 observations. B: steady-state inactivation of INa shifted by 10 nM T3 while 100 µM GTP (triangle ) or GTPgamma S () was present in the pipette solution. The currents were elicited with preconditioning pulse of 500-ms duration from -150 to -50 mV to a test potential of +20 mV. The normalized INa plotted against preconditioning pulse potential was fitted using a Boltzmann equation.

T3 effects on single INa in cell-attached patch experiments. In considering the slow onset of T3 effect, the time dependence of the single INa activity was examined under control conditions. It seemed that traces recorded at 0-5 min have less null sweeps and more openings. The peak of ensemble currents recorded at 0-5 min was significantly greater than that recorded at 5-10 (P < 0.005), 15-20 (P < 0.01), and 25-45 min (P < 0.05). There was no significant change in the peak of ensemble currents during 5-45 min. Within 5 min after the formation of gigaohm seals, the changes varied; 7 of 10 patches showed a decrease, 1 of 10 patches showed an increase, and 2 of 10 patches showed no change. The unit amplitude did not change while recording. The number of openings decreased by 8% in 5 min and thereafter remained constant. The mean open time (to) gradually increased by 12%, and PN gradually increased by 17%, reaching a plateau in 5 min, but these changes were not statistically significant. These results suggest that single INa was not stable within the first 5 min and relatively stable over the 5-45 min after the formation of gigaohm seals under our experimental conditions. Therefore, we recorded all data for the estimation of T3 effects on single INa during a period of 5-30 min after the formation of gigaohm seals.

Sixteen consecutive sweeps at VT = -50 mV in control and 25 min after superfusion with 10 nM T3 are shown in Fig. 4A. This patch had three channels. After superfusion with T3, the number of null sweeps was decreased and the number of openings was increased. The unit amplitude was unchanged after T3 superfusion. Figure 4B shows the change in the ensemble currents by T3. The peak of the ensemble currents with T3 was significantly greater than the peak without T3 at VT = -60 to -30 mV. At VT = -30 mV, the percent change in the peak of the ensemble currents induced by T3 was 45% (n = 8, P < 0.005) and was similar to that seen in the whole cell recordings (Fig. 4C). As shown in Fig. 5A, PN was 0.4-0.7 in controls and the relation between PN and the membrane potential was V shaped with a turning point at VT = -40 mV. The value of PN was the same as that previously reported (38). T3 reduced PN significantly at VT = -60 to -30 mV by 16-43%. No increase in the number of available channels was observed while superfusing with T3. In controls, to was 0.2-0.8 ms at VT = -60 to -20 mV and longer at more positive membrane potentials but showed minimal voltage dependence over a range of VT = -30 mV (Fig. 5B). This tendency is consistent with previous reports, but the values of to were shorter than that previously reported (0.3-3.0 ms) (24, 26, 38). Shorter to is considered to be due to a little higher experimental temperature (22-24°C) and/or shorter sampling interval (50 µs). The values and the tendency against the membrane potential were not altered by T3. The number of openings was 1.3-1.7 and increased at more negative membrane potentials in controls (35). T3 did not change to or the number of openings at VT = -60 to -30 mV (Fig. 6, A and B). The relation between the unit amplitude and the membrane potential was shown in Fig. 6C. The slope conductance was 5.3 pS in control and 5.2 pS 25 min after superfusion with T3. The effects of T3 on Po were also evaluated. Figure 7A demonstrated the Po in a test pulse duration of 50 ms in control solution and 25 after superfusion with 10 nM T3 at VT = -30 mV. T3 increased the peak Po from 8 to 16%. The effects of T3 on the peak Po were plotted against the membrane potential in Fig. 7B. The peak Po was significantly increased at VT = -60 to -30 mV (57% at VT = -30 mV, n = 8, P < 0.005).


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  		Fig. 4.   A: consecutive current traces at VT = -50 mV in control and 25 min after superfusing with 10 nM T3. This patch had three channels. N represents a null sweep. Holding potential was -140 mV. B: changes in ensemble currents by T3. Ensemble current tracings at VT = -30 mV in control and 25 min after being superfused with 10 nM T3. Holding potential was -140 mV. C: changes in the peak of ensemble currents by 10 nM T3 at various test potentials. open circle , Data recorded in control; , data recorded 25 min after superfusion with 1 nM T3. The number of data with and without T3 were 6 at VT = -60 mV, 7 at VT = -50 mV, 7 at VT = -40 mV, and 8 at VT = -30 mV. * P < 0.05; ** P < 0.005 vs. control.



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  		Fig. 5.   A: changes in null probability by 10 nM T3 at various test potentials in the different patch experiments. , Data recorded in control; black-down-triangle , data recorded 25 min after superfusion with 10 nM T3. * P < 0.05; ** P < 0.005; *** P < 0.001 vs. control. B: open time distribution histograms in control and 25 after superfusion with 1 nM T3 at VT = -60, -50, and -40 mV. Bin width was 0.1 ms. The first bin was omitted. The number of total events in control was 361, 989, and 1,481 at VT = -60, -50, and -40 mV, respectively. The number of total events after superfusion with T3 was 661, 1,159, and 868 at VT = -60, -50, and -40 mV, respectively. There was no missing value. The unit of time constants (tau o) was in milliseconds.



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  		Fig. 6.   Effects of 10 nM T3 on mean open time (A), number of openings (B), and unit amplitude (C) of single INa. open circle , Data recorded in control; , data recorded 25 min after superfusion with 10 nM T3. There was no statistical significance (NS). In C, the slope conductance was 5.3 pS in control and 5.2 pS after superfusion with T3 (NS). swe, Sweep.



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  		Fig. 7.   A: changes in open probability by 10 nM T3. The tracing of open probability at VT = -50 mV recorded in control was shown on the left and 25 min after superfusion with T3 was shown on the right. B: changes in peak open probability by T3 at various test potentials. Open bars represent the data recorded in control and solid bars represent the data recorded 25 min after superfusion with T3. * P < 0.05; ** P < 0.005 vs. control.

T3 effects on single INa in inside-out patch experiments. Figure 8A demonstrates 16 consecutive sweeps and the ensemble currents before and 25 min after superfusion with 1 nM T3 and 100 µM GTPgamma S at VT = -40 mV in an inside-out patch configuration containing two channels. Similar to our results in the cell-attached patch experiments, T3 decreased the number of null sweeps and increased the peak of the ensemble currents. As shown in Table 1, the peak of the ensemble currents increased 64% by 1 nM T3 (P < 0.01), PN was reduced 35% (P < 0.01), and to was slightly increased (P = 0.052) at VT = -40 mV. The effect of 1 nM T3 with 100 µM GTPgamma S on the peak of the ensemble currents was 39.7 ± 8.7% greater compared with cell-attached patch experiments (P < 0.01). However, the effects on PN were quantitatively the same as that in cell-attached patch experiments. To elucidate the detailed mechanism, we assumed a five-state model, which is given by the scheme
where R1, R2, and C are closed, rested states, O is an open state, I is a closed, absorbing inactivated state, and the rate constants kCO, kOC, kCI, and kOI represent closed state to open state, open state to closed state, closed state to absorbing inactivated state, and open state to absorbing inactivated state, respectively (38). As shown in Table 1, 1 nM T3 with 100 µM GTPgamma S significantly increased kCO for the transition from the C to O state. kOI, for the transition from the O to I state, was significantly decreased by T3. However, the other rate constants were not significantly changed at any membrane potential tested.


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  		Fig. 8.   A: consecutive current traces and ensemble currents (bottom traces) at VT = -40 mV before and 25 min after superfusion with 1 nM T3 associated with 100 µM GTPgamma S in the same inside-out patch. This patch had two channels. N, null sweep. The dashed line represents zero level for ensemble currents. Holding potential was -140 mV. B: time course of T3 effect on the open probability of single INa recorded by inside-out patch.


                              
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  		Table 1.   Effects of T3 on single INa in the inside-out patches

Figure 8B shows a reproducible inside-out patch recording from a cell sequentially treated with T3, GTPgamma S, and then with T3 and GTPgamma S together. With no GTP or GTPgamma S present, application of 10 nM T3 onto the cytoplasmic surface of the cell membrane only slightly increased the Po of single INa (17 ± 4%, P < 0.05) by reducing PN (P < 0.05). The rate constant, kCO, was only slightly increased (8 ± 2%, P = 0.043), and kOI was slightly decreased (4 ± 1%), but this trend did not reach statistical significance (P = 0.052). These effects could be completely washed out. GTPgamma S (10 µM) alone significantly increased the peak of ensemble current 48.9 ± 12.2% (n = 25, P < 0.01) and slightly increased the Po of the single INa. GTPgamma S increased the number of functional channels in 7 of 15 patches with two channels, but did not alter the single INa amplitude, to, PN, or the channel kinetics. ATP was not present in the bath solution, suggesting that the effects of GTPgamma S were not mediated by activating adenylate cyclase within the patch (23). In the same patch, when 10 µM GTPgamma S was present, the Po of single INa was immediately increased by the addition of 10 nM T3 onto the cytoplasmic side of the membrane. This effect was observed within 30 s after T3 exposure and reached a plateau within 3 min. T3 and GTPgamma S synergistically increased the Po by 5.8 ± 0.5-fold, and this effect was much greater than that seen in whole cell and cell-attached experiments. PN was reduced 91 ± 3% compared with that in control conditions, but single INa amplitude was unchanged. The rate constant kCO increased 3.2 ± 0.7-fold, and kOI was significantly decreased (18 ± 4%, P < 0.01). The increase in INa was reversible on T3 and GTPgamma S washout.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

T3 activates INa through extranuclear mechanism. Our results provide direct evidence that T3 produces a dose-dependent increase in INa in adult ventricular myocytes through an extranuclear mechanism. Although a T3 receptor on the plasma membrane of cardiac myoyctes has not been structurally characterized, numerous studies have suggested its functional existence. The onset of T3 effects on INa was evident within 5-15 min in whole cell and cell-attached patch experiments. The time course of the T3 effect on INa is consistent with that on K+ or Ca2+ channel current in adult cardiac myocytes (19, 33, 37). In neonatal myocytes, however, the effect of T3 on INa occurred relatively faster, although the changing of current density and the shifting of the inactivation curve were similar to adult ventricular myocytes (20). This difference might result from the different membrane permeability to thyroid hormone in the neonatal myocytes compared with adult myocytes. The time frame of T3 effect on ion channels is considered to be too short to express specific genes within the nucleus of the cell (39). The same amount of enhancement in INa current was induced by TAA, an analog of T3 that was considered, does not induce DNA transcription in the nucleus (37). However, recent studies (1, 29) suggest TAA also has a transcriptional effect. In the present study, we used an inside-out patch technique to examine the effects of T3 on the INa in the plasma membrane. For the first time, we have been able to provide direct evidence that T3 acts on the cytoplasmic side of the plasma membrane and activates the INa through an extranuclear mechanism. This explains the relatively slow action of T3 observed in the whole cell and cell-attached patch experiments reported previously by our laboratory and other groups (19, 20, 33, 37). In one exception, Dudley et al. (7) found a significant increase in the single INa bursting in the cell-attached patches with T3 in the pipette solution. In that study, the comparison between control and T3 groups was made in different patches. The bursting occurred less than a millisecond after patch excision. Concern has been raised that the recording was made immediately after the patch formed, which is in the unstable period for parameters of single INa. This data could be affected by time-dependent kinetic changes of single INa and their variability (12, 17). Thus the observations may be hardly distinguishable from artifacts of the patch maneuver in both cell-attached patch and inside-out patch reported previously (23, 24). On the other hand, the high tension on the patched membrane might facilitate the T3 in the pipette to permeate into the membrane and promote the fast action of T3 on the channel.

G protein modulates the T3 effect on INa. Our results demonstrate that a pathophysiological concentration of T3 acts directly on the cytosolic side of the membrane and weakly, but significantly activates INa. The synergism of a pathophysiological concentration of pertussis toxin-sensitive G protein and T3 greatly magnifies the effects of T3. In a whole cell configuration with GTP-free pipette solution, 10 nM T3 increased the peak INa 47%, and slightly shifted the inactivation curve. With the addition of 100 µM GTP or GTPgamma S into the pipette solution, the effects of T3 on the peak I-V curve of INa and the shifting inactivation curve were shifted more than two fold. Pertussis toxin greatly reduced, but did not completely abolish, the effect of T3, suggesting a direct effect of T3 on INa. Because most G proteins involve channel activation, the effects of T3 on INa were also more prominent at hyperpolarized potentials (27, 28). The rightward shift of the steady-state inactivation curve seen in this study was similar to that observed in other hormonal effects which involve the second messenger or signal transducing components (24). Nevertheless, previous studies (27, 28) have shown that the direct effects of G protein on INa are not associated with changes in inactivation kinetics. Therefore, T3 was responsible for the change in the inactivation kinetics. In cell-attached configurations, the physiological concentration of GTP was preserved. T3 effects were initiated earlier than that in whole cell recordings with GTP-free medium, but still later than that in the whole cell patches with a saturating concentration of GTP in the medium. In the inside-out configuration, without GTPgamma S present, T3 only slightly increased the single INa Po, but this effect was initiated 10 times faster than that in whole cell and cell attached configurations. This effect could be washed out completely in the inside-out configuration. These observations provide direct evidence that T3 acts on the cytosolic side of the membrane and directly activates INa. In the presence of the GTPgamma S, T3 greatly increased the Po of the channel within 1 min and reached a maximum within 3 min. This time course was consistent with other G protein-involved channel regulation, such as isoproterenol (28). The strong interaction of the channel with a pathophysiological concentration of T3 in the presence of GTPgamma S suggests that G proteins might induce a conformational change that has a profound impact on the interaction of the channel with T3. Petit-Jacques et al. (35) reported the synergistic activation of muscarinic K+ channel by G proteins and Na+ and Mg2+. Our findings imply that variations in the local levels of G protein may have great impact on the interaction of a pathophysiological concentration of T3 with INa in ventricular myocytes.

Detailed mechanism of T3 effects on kinetics of single INa. The present study demonstrates that T3 increased the Po of single INa by reducing the PN without changing to or the number of available channels. Horn et al. (21) proposed that INa can inactivate without opening. Nulls are considered to occur by inactivation of the channel directly from the closed state without entering the open state. However, Scanley et al. (38) mentioned four alternative possibilities that could cause overestimation and at least one that would cause underestimation of PN: 1) an opening occurred that was too short to be detected (2), 2) an early opening occurred that was obscured by the capacity transient, 3) channels were already in the inactivated state at the time of the step, or 4) channels remaining in a closed, noninactivated state throughout the step (31). PN is reported to be higher at more negative potentials (31). However, in this study, the relation between the membrane potential and PN was V shaped. At positive potentials, such as -30 mV, waiting time to the first opening was very short and reopening was rare. Therefore, the capacitive transient could obscure early openings at VT = -30 mV more than those at more negative membrane potentials and PN could be overestimated. In a basic five-state model for single INa, PN is kCI/(kCI + kCO) (38). As we reported previously, T3 accelerated the transition from a deep closed state to a shallow closed state in a single inward rectifier K+ channel using a three-state model (C1-C2-O) (31). In the present study, T3 also significantly increases kCO, accelerates the transition, and decreases PN. Scanley et al. (38) mentioned that the relation between PN and the membrane potential showed a negative shift at warmer temperatures because the kCO transition rate could be more sensitive to temperature than the kCI rate. This suggests that the sensor to temperature effects in INa might be same as that to thyroid hormonal effects.

Most prior reports on to for cardiac single INa have indicated that the distribution is well fitted to a single exponential function (12, 32), although some investigators have reported a two exponential distribution (26). Burst type openings have been reported to be different from nonburst type openings (9, 25, 31). The time constant of to in bursts is longer (3-5 times) than that of nonburst type openings. This suggests that the single INa have at least two open states. However, the incidence of burst type openings is rare (<1%) (9, 25, 31). In this study, its incidence was <0.3% and was not altered by T3. Therefore, the burst type openings were excluded from analysis.

In a single open state, to is described by the inverse of the sum of rate constants leaving the open state. Therefore, in a basic five-state model, to could be calculated as (kOC + kOI)-1. In cell-attached patches, a physiological concentration of G protein remained. The addition of T3 extracellularly did not change to at VT = -60 to -20 mV, suggesting that T3 does not significantly change the sum of kOC and kOI. In inside-out patches, the increase of kCO and the decrease of kOI induced by a pathophysiological concentration of T3 were revealed, and these effects were independent of G protein. The presence of a pathophysiological concentration of GTPgamma S and T3 resulted in a stimulation of channel activity that was much greater than the simple sum of their individual effects. G protein increases the number of functional channels, which could have impact on the effect of T3 on the channel Po (27). However, this does not fully explain the accelerated effects of T3 on channel kinetics in the presence of G protein. It is more likely that T3 and G protein exert their combined effects by synergistic interaction of different channel sites. G protein induces channel conformational changes that may sensitize the channel for T3-induced alterations in channel kinetics, thereby reducing PN and increasing the channel Po. The increased number of functional channels by G protein might further magnify the action of T3 on the channel open probability.

Physiological significance. The upstroke velocity of the action potential (Vmax) is attributed to INa currents and there is a controversy as to whether thyroid hormones can affect Vmax or not. A previous investigation has reported that Vmax did not change in hyperthyroid rabbit papillary muscle (42). Acutely administered T3 has been reported to show no effect on Vmax in guinea pig ventricular myocytes (10). However, Freedberg et al. (14) reported that Vmax was increased in hyperthyroidism and decreased in hypothyroidism at certain driving frequencies in rabbit atrial cells. Johnson et al. (22) also demonstrated the increase in Vmax in hyperthyroidism in rabbit atrial cells. Enhanced INa by T3, which could result in an increased Vmax, would therefore increase the conduction rate and contractility. However, this effect may be more pronounced in the atrial cells than ventricular cells. The reason for this difference is not clear, it could be due to T3 penetrates the membrane of atrial cells easier than ventricular cells, and/or atrial cells responses to T3 better than ventricular cells. On the other hand, a synergistic effect of G protein and T3 would significantly decrease the kOI. Relatively higher inhibitory Galpha (Galpha i) in the atrial cells may also responsible for the amplified T3 effect in the atrium.

Recent studies (8) have shown that slowing INa inactivation prolongs the QT interval and aggravates adrenaline-induced arrhythmias. The incidence of ventricular arrhythmias in hyperthyroidism is much lower than supraventricular arrhythmias (44). However, hyperthyroid patients with cardiac hypertrophy and heart failure were generally not included in these studies (44) because ventricular arrhythmias in this patient population are not considered to be related to hyperthyroidism. Substantial increases in Galpha i in the setting of hypertrophy, ischemic cardiomyopathy, and heart failure have been found both in animal models and in patients (11). Although pathophysiologically elevated T3 level may have only minor effect on the ion channels. The synergistic interaction between the pathophysiologically increased G protein and T3 levels may have a profound impact on Na+ channel kinetics. This synergy significantly slows Na+ channel inactivation, increases fast inward INa, and consequently increases the membrane excitability that may be the cofactor of ventricular arrhythmias in hyperthyroid patients with hypertrophy, ischemia, and/or heart failure (12).


    ACKNOWLEDGEMENTS

We thank Dr. Bramah H. Singh for support, Dr. Paul J. Davis for discussions, and Di-Chong Xu for help.


    FOOTNOTES

This study was partially supported by grants from the American Heart Association, Greater Los Angeles Affiliate, and the Laubish Fund, University of California Regents.

Address for reprint requests and other correspondence: L. Sen, Div. of Cardiology, UCLA Medical Center, The David Geffen School of Medicine, 47-123 CHS, 10833 Le Conte Ave., Los Angeles, CA 90095-1679 (E-mail: lsen{at}mednet.ucla.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

July 26, 2002;10.1152/ajpheart.00326.2002

Received 10 April 2002; accepted in final form 24 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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