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Channel openings are necessary but not sufficient for use-dependent block of cardiac Na(+) channels by flecainide: evidence from the analysis of disease-linked mutations.

Liu H, Tateyama M, Clancy CE, Abriel H, Kass RS - J. Gen. Physiol. (2002)

Bottom Line: Na(+) channel blockers such as flecainide have found renewed usefulness in the diagnosis and treatment of two clinical syndromes arising from inherited mutations in SCN5A, the gene encoding the alpha subunit of the cardiac voltage-gated Na(+) channel.Our results indicate that mutation-induced changes in the voltage-dependence of channel availability (inactivation) may act as determinants of flecainide block.Analysis of flecainide block of mutant channels linked to these rare disorders has provided novel insight into the molecular determinants of drug action.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA.

ABSTRACT
Na(+) channel blockers such as flecainide have found renewed usefulness in the diagnosis and treatment of two clinical syndromes arising from inherited mutations in SCN5A, the gene encoding the alpha subunit of the cardiac voltage-gated Na(+) channel. The Brugada syndrome (BrS) and the LQT-3 variant of the Long QT syndrome are caused by disease-linked SCN5A mutations that act to change functional and pharmacological properties of the channel. Here we have explored a set of SCN5A mutations linked both to BrS and LQT-3 to determine what disease-modified channel properties underlie distinct responses to the Na(+) channel blocker flecainide. We focused on flecainide block that develops with repetitive channel activity, so-called use-dependent block (UDB). Our results indicate that mutation-induced changes in the voltage-dependence of channel availability (inactivation) may act as determinants of flecainide block. The data further indicate that UDB by flecainide requires channel opening, but is not likely due to open channel block. Rather, flecainide appears to interact with inactivation states that follow depolarization-induced channel opening, and mutation-induced changes in channel inactivation will alter flecainide block independent of the disease to which the mutation is linked. Analysis of flecainide block of mutant channels linked to these rare disorders has provided novel insight into the molecular determinants of drug action.

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Single channel and averaged ensemble current properties of WT and mutant channels. Cell-attached patch recordings are shown for WT, D1709G (DG), Y1795H (YH), and Y1795C (YC) channels. Recordings were obtained in response to test pulses (−30 mV, 100 ms) applied at 2 Hz from −120 mV. (A) Current recordings of individual and consecutive sweeps are shown to emphasize the effects of inherited mutations on channel opening kinetics. Ensemble currents (constructed by averaging 500 consecutive sweeps) are shown for each construct below the individual sweeps. (B) Open time distributions of WT and mutants channels. Open time histograms were generated for each construct using 200-μs bins of all events recorded from 500–1,000 sweeps. Patches used included three or fewer channels. MOT was estimated by the best-fit single exponential function to each histogram. The fitted curves are illustrated as dashed lines in each panel. The resulting MOT estimates are: 0.50 ± 0.02 ms (WT); 0.42 ± 0.03 ms (DG); 0.39 ± 0.02 ms (YH); and 0.97 ± 0.05 ms (YC); P < 0.05 for DG, YH, and YC vs. WT, respectively, n = 6–7 patches per condition. (C) Time course of the onset of UDB (1 Hz, 10 μM flecainide) during pulse trains applied to WT and YC channels. The data were normalized to the current amplitude of the first pulse in the train and fit with a single exponential function (A × exp-t/τ + base), the time constant (τ) for WT and Y1795C were 45.29 s−1 and 20.09 s−1 (P < 0.01 vs. WT, n = 3 cells per condition).
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fig4: Single channel and averaged ensemble current properties of WT and mutant channels. Cell-attached patch recordings are shown for WT, D1709G (DG), Y1795H (YH), and Y1795C (YC) channels. Recordings were obtained in response to test pulses (−30 mV, 100 ms) applied at 2 Hz from −120 mV. (A) Current recordings of individual and consecutive sweeps are shown to emphasize the effects of inherited mutations on channel opening kinetics. Ensemble currents (constructed by averaging 500 consecutive sweeps) are shown for each construct below the individual sweeps. (B) Open time distributions of WT and mutants channels. Open time histograms were generated for each construct using 200-μs bins of all events recorded from 500–1,000 sweeps. Patches used included three or fewer channels. MOT was estimated by the best-fit single exponential function to each histogram. The fitted curves are illustrated as dashed lines in each panel. The resulting MOT estimates are: 0.50 ± 0.02 ms (WT); 0.42 ± 0.03 ms (DG); 0.39 ± 0.02 ms (YH); and 0.97 ± 0.05 ms (YC); P < 0.05 for DG, YH, and YC vs. WT, respectively, n = 6–7 patches per condition. (C) Time course of the onset of UDB (1 Hz, 10 μM flecainide) during pulse trains applied to WT and YC channels. The data were normalized to the current amplitude of the first pulse in the train and fit with a single exponential function (A × exp-t/τ + base), the time constant (τ) for WT and Y1795C were 45.29 s−1 and 20.09 s−1 (P < 0.01 vs. WT, n = 3 cells per condition).

Mentions: Fig. 2 illustrates the effect of COOH-terminal mutations on UDB, whereas Fig. 3 demonstrates the necessity of channel opening for the development of this block. To rule out the possibility that flecainide UDB is due to block of open channels, we tested the effect of channel mean open time (MOT) on drug affinity. If flecainide blocks open channels, then it follows that mutation-induced changes in MOTs would be expected to parallel mutation-induced changes in UDB (Anno and Hondeghem, 1990). Channel mean open times are significantly shorter for D1790G and Y1795H, and significantly longer for Y1795C channels compared with WT channels (Fig. 4, A and B). If flecainide were to block open channels, we would expect that D1790G and Y1795H would exhibit less UDB than Y1795C channels. However, we find the opposite: Y1795C are less sensitive to flecainide UDB than either Y1795H or D1790G channels. Together, these results strongly suggest that flecainide requires channels to open first in order to bind, but is not dependent on the open state to promote block. This possibility is further supported by the data presented in Fig. 4 C, which compares the time course of the onset of block for WT and Y1795C channels. Here the development of block is clearly faster for Y1795C channels, but the steady-state block is the same as WT. This effect might be expected if entry through open channels is a prerequisite for block, but that affinity of block is not determined by the open state of the channel. Our data raise another interesting possibility that the resulting inactivated state may be playing an important role in flecainide block.


Channel openings are necessary but not sufficient for use-dependent block of cardiac Na(+) channels by flecainide: evidence from the analysis of disease-linked mutations.

Liu H, Tateyama M, Clancy CE, Abriel H, Kass RS - J. Gen. Physiol. (2002)

Single channel and averaged ensemble current properties of WT and mutant channels. Cell-attached patch recordings are shown for WT, D1709G (DG), Y1795H (YH), and Y1795C (YC) channels. Recordings were obtained in response to test pulses (−30 mV, 100 ms) applied at 2 Hz from −120 mV. (A) Current recordings of individual and consecutive sweeps are shown to emphasize the effects of inherited mutations on channel opening kinetics. Ensemble currents (constructed by averaging 500 consecutive sweeps) are shown for each construct below the individual sweeps. (B) Open time distributions of WT and mutants channels. Open time histograms were generated for each construct using 200-μs bins of all events recorded from 500–1,000 sweeps. Patches used included three or fewer channels. MOT was estimated by the best-fit single exponential function to each histogram. The fitted curves are illustrated as dashed lines in each panel. The resulting MOT estimates are: 0.50 ± 0.02 ms (WT); 0.42 ± 0.03 ms (DG); 0.39 ± 0.02 ms (YH); and 0.97 ± 0.05 ms (YC); P < 0.05 for DG, YH, and YC vs. WT, respectively, n = 6–7 patches per condition. (C) Time course of the onset of UDB (1 Hz, 10 μM flecainide) during pulse trains applied to WT and YC channels. The data were normalized to the current amplitude of the first pulse in the train and fit with a single exponential function (A × exp-t/τ + base), the time constant (τ) for WT and Y1795C were 45.29 s−1 and 20.09 s−1 (P < 0.01 vs. WT, n = 3 cells per condition).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2311398&req=5

fig4: Single channel and averaged ensemble current properties of WT and mutant channels. Cell-attached patch recordings are shown for WT, D1709G (DG), Y1795H (YH), and Y1795C (YC) channels. Recordings were obtained in response to test pulses (−30 mV, 100 ms) applied at 2 Hz from −120 mV. (A) Current recordings of individual and consecutive sweeps are shown to emphasize the effects of inherited mutations on channel opening kinetics. Ensemble currents (constructed by averaging 500 consecutive sweeps) are shown for each construct below the individual sweeps. (B) Open time distributions of WT and mutants channels. Open time histograms were generated for each construct using 200-μs bins of all events recorded from 500–1,000 sweeps. Patches used included three or fewer channels. MOT was estimated by the best-fit single exponential function to each histogram. The fitted curves are illustrated as dashed lines in each panel. The resulting MOT estimates are: 0.50 ± 0.02 ms (WT); 0.42 ± 0.03 ms (DG); 0.39 ± 0.02 ms (YH); and 0.97 ± 0.05 ms (YC); P < 0.05 for DG, YH, and YC vs. WT, respectively, n = 6–7 patches per condition. (C) Time course of the onset of UDB (1 Hz, 10 μM flecainide) during pulse trains applied to WT and YC channels. The data were normalized to the current amplitude of the first pulse in the train and fit with a single exponential function (A × exp-t/τ + base), the time constant (τ) for WT and Y1795C were 45.29 s−1 and 20.09 s−1 (P < 0.01 vs. WT, n = 3 cells per condition).
Mentions: Fig. 2 illustrates the effect of COOH-terminal mutations on UDB, whereas Fig. 3 demonstrates the necessity of channel opening for the development of this block. To rule out the possibility that flecainide UDB is due to block of open channels, we tested the effect of channel mean open time (MOT) on drug affinity. If flecainide blocks open channels, then it follows that mutation-induced changes in MOTs would be expected to parallel mutation-induced changes in UDB (Anno and Hondeghem, 1990). Channel mean open times are significantly shorter for D1790G and Y1795H, and significantly longer for Y1795C channels compared with WT channels (Fig. 4, A and B). If flecainide were to block open channels, we would expect that D1790G and Y1795H would exhibit less UDB than Y1795C channels. However, we find the opposite: Y1795C are less sensitive to flecainide UDB than either Y1795H or D1790G channels. Together, these results strongly suggest that flecainide requires channels to open first in order to bind, but is not dependent on the open state to promote block. This possibility is further supported by the data presented in Fig. 4 C, which compares the time course of the onset of block for WT and Y1795C channels. Here the development of block is clearly faster for Y1795C channels, but the steady-state block is the same as WT. This effect might be expected if entry through open channels is a prerequisite for block, but that affinity of block is not determined by the open state of the channel. Our data raise another interesting possibility that the resulting inactivated state may be playing an important role in flecainide block.

Bottom Line: Na(+) channel blockers such as flecainide have found renewed usefulness in the diagnosis and treatment of two clinical syndromes arising from inherited mutations in SCN5A, the gene encoding the alpha subunit of the cardiac voltage-gated Na(+) channel.Our results indicate that mutation-induced changes in the voltage-dependence of channel availability (inactivation) may act as determinants of flecainide block.Analysis of flecainide block of mutant channels linked to these rare disorders has provided novel insight into the molecular determinants of drug action.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA.

ABSTRACT
Na(+) channel blockers such as flecainide have found renewed usefulness in the diagnosis and treatment of two clinical syndromes arising from inherited mutations in SCN5A, the gene encoding the alpha subunit of the cardiac voltage-gated Na(+) channel. The Brugada syndrome (BrS) and the LQT-3 variant of the Long QT syndrome are caused by disease-linked SCN5A mutations that act to change functional and pharmacological properties of the channel. Here we have explored a set of SCN5A mutations linked both to BrS and LQT-3 to determine what disease-modified channel properties underlie distinct responses to the Na(+) channel blocker flecainide. We focused on flecainide block that develops with repetitive channel activity, so-called use-dependent block (UDB). Our results indicate that mutation-induced changes in the voltage-dependence of channel availability (inactivation) may act as determinants of flecainide block. The data further indicate that UDB by flecainide requires channel opening, but is not likely due to open channel block. Rather, flecainide appears to interact with inactivation states that follow depolarization-induced channel opening, and mutation-induced changes in channel inactivation will alter flecainide block independent of the disease to which the mutation is linked. Analysis of flecainide block of mutant channels linked to these rare disorders has provided novel insight into the molecular determinants of drug action.

Show MeSH
Related in: MedlinePlus