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Current-dependent block of rabbit sino-atrial node I(f) channels by ivabradine.

Bucchi A, Baruscotti M, DiFrancesco D - J. Gen. Physiol. (2002)

Bottom Line: In this, the action of ivabradine on f-channels is similar to that reported of other rate-reducing agents such as UL-FS49 and ZD7288.Bound drug molecules do not detach from the binding site in the absence of inward current through channels, even if channels are open and the drug is therefore not "trapped" by closed gates.The use-dependence resulting from specific features of I(f) block by ivabradine amplifies its rate-reducing ability at high spontaneous rates and may be useful to clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Department of General Physiology and Biochemistry, Laboratory of Molecular Physiology and Neurobiology, and INFM-Unità Milano Università, 20133 Milano, Italy.

ABSTRACT
"Funny" (f-) channels have a key role in generation of spontaneous activity of pacemaker cells and mediate autonomic control of cardiac rate; f-channels and the related neuronal h-channels are composed of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel subunits. We have investigated the block of f-channels of rabbit cardiac sino-atrial node cells by ivabradine, a novel heart rate-reducing agent. Ivabradine is an open-channel blocker; however, block is exerted preferentially when channels deactivate on depolarization, and is relieved by long hyperpolarizing steps. These features give rise to use-dependent behavior. In this, the action of ivabradine on f-channels is similar to that reported of other rate-reducing agents such as UL-FS49 and ZD7288. However, other features of ivabradine-induced block are peculiar and do not comply with the hypothesis that the voltage-dependence of block is entirely attributable to either the sensitivity of ivabradine-charged molecules to the electrical field in the channel pore, or to differential affinity to different channel states, as has been proposed for UL-FS49 (DiFrancesco, D. 1994. Pflugers Arch. 427:64-70) and ZD7288 (Shin, S.K., B.S. Rotheberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91-101), respectively. Experiments where current flows through channels is modified without changing membrane voltage reveal that the ivabradine block depends on the current driving force, rather than voltage alone, a feature typical of block induced in inwardly rectifying K(+) channels by intracellular cations. Bound drug molecules do not detach from the binding site in the absence of inward current through channels, even if channels are open and the drug is therefore not "trapped" by closed gates. Our data suggest that permeation through f-channel pores occurs according to a multiion, single-file mechanism, and that block/unblock by ivabradine is coupled to ionic flow. The use-dependence resulting from specific features of I(f) block by ivabradine amplifies its rate-reducing ability at high spontaneous rates and may be useful to clinical applications.

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Hyperpolarization favors removal of block. A long (40 s) hyperpolarizing step to −100 mV was preceded and followed by a repetitive activation/deactivation protocol (−100/+5 mV) during perfusion with ivabradine (3 μM). (A) Left to right: current traces recorded in control (cont) and 30 (a), 60 (b), and 174 s (c) after switching on of drug perfusion; current record during the 40 s step (d); current traces recorded just after termination of the 40 s step (e) and 30 (f), 60 (g), and 90 s later (h). Note that If increased slowly during the long −100 mV step; the inset shows a superimposition of traces c and e. (B) Semilog plot of the first 10 s of the long step record; fitting with the sum of two exponentials (broken lines) yielded a fast and a slow component (time constant values in text).
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fig4: Hyperpolarization favors removal of block. A long (40 s) hyperpolarizing step to −100 mV was preceded and followed by a repetitive activation/deactivation protocol (−100/+5 mV) during perfusion with ivabradine (3 μM). (A) Left to right: current traces recorded in control (cont) and 30 (a), 60 (b), and 174 s (c) after switching on of drug perfusion; current record during the 40 s step (d); current traces recorded just after termination of the 40 s step (e) and 30 (f), 60 (g), and 90 s later (h). Note that If increased slowly during the long −100 mV step; the inset shows a superimposition of traces c and e. (B) Semilog plot of the first 10 s of the long step record; fitting with the sum of two exponentials (broken lines) yielded a fast and a slow component (time constant values in text).

Mentions: To verify the presence of hyperpolarization-induced block relief, we used the analysis shown in Fig. 4. A standard activation/deactivation protocol was first applied and the drug (3 μM) was perfused until steady-state block developed. On the left side of Fig. 4 A the control trace and traces recorded after 30, 60, and 174 s (corresponding to steady-state block) of drug perfusion are plotted in sequence. Once steady-state block was achieved, while still in the presence of the drug, a long (40 s) step to –100 mV was applied, during which the current underwent a gradual, slow increase with time toward levels approaching that in control conditions. A double exponential fit of the current during the 40-s long step (Fig. 4 B) clearly revealed the presence of two kinetically distinct processes. Although the early part of current activation developed with a time constant of 235 ms, a value similar to that in control conditions (259 ms), the late one increased with a much slower time constant of 7.5 s; this second process did not therefore reflect normal activation kinetics, but rather removal of block possibly associated with unbinding of the drug. Resuming the activation/deactivation protocol reestablished the previous current block, as apparent from the set of current traces recorded every 30 s after the long step to −100 mV (right side of Fig. 4 A).


Current-dependent block of rabbit sino-atrial node I(f) channels by ivabradine.

Bucchi A, Baruscotti M, DiFrancesco D - J. Gen. Physiol. (2002)

Hyperpolarization favors removal of block. A long (40 s) hyperpolarizing step to −100 mV was preceded and followed by a repetitive activation/deactivation protocol (−100/+5 mV) during perfusion with ivabradine (3 μM). (A) Left to right: current traces recorded in control (cont) and 30 (a), 60 (b), and 174 s (c) after switching on of drug perfusion; current record during the 40 s step (d); current traces recorded just after termination of the 40 s step (e) and 30 (f), 60 (g), and 90 s later (h). Note that If increased slowly during the long −100 mV step; the inset shows a superimposition of traces c and e. (B) Semilog plot of the first 10 s of the long step record; fitting with the sum of two exponentials (broken lines) yielded a fast and a slow component (time constant values in text).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2238187&req=5

fig4: Hyperpolarization favors removal of block. A long (40 s) hyperpolarizing step to −100 mV was preceded and followed by a repetitive activation/deactivation protocol (−100/+5 mV) during perfusion with ivabradine (3 μM). (A) Left to right: current traces recorded in control (cont) and 30 (a), 60 (b), and 174 s (c) after switching on of drug perfusion; current record during the 40 s step (d); current traces recorded just after termination of the 40 s step (e) and 30 (f), 60 (g), and 90 s later (h). Note that If increased slowly during the long −100 mV step; the inset shows a superimposition of traces c and e. (B) Semilog plot of the first 10 s of the long step record; fitting with the sum of two exponentials (broken lines) yielded a fast and a slow component (time constant values in text).
Mentions: To verify the presence of hyperpolarization-induced block relief, we used the analysis shown in Fig. 4. A standard activation/deactivation protocol was first applied and the drug (3 μM) was perfused until steady-state block developed. On the left side of Fig. 4 A the control trace and traces recorded after 30, 60, and 174 s (corresponding to steady-state block) of drug perfusion are plotted in sequence. Once steady-state block was achieved, while still in the presence of the drug, a long (40 s) step to –100 mV was applied, during which the current underwent a gradual, slow increase with time toward levels approaching that in control conditions. A double exponential fit of the current during the 40-s long step (Fig. 4 B) clearly revealed the presence of two kinetically distinct processes. Although the early part of current activation developed with a time constant of 235 ms, a value similar to that in control conditions (259 ms), the late one increased with a much slower time constant of 7.5 s; this second process did not therefore reflect normal activation kinetics, but rather removal of block possibly associated with unbinding of the drug. Resuming the activation/deactivation protocol reestablished the previous current block, as apparent from the set of current traces recorded every 30 s after the long step to −100 mV (right side of Fig. 4 A).

Bottom Line: In this, the action of ivabradine on f-channels is similar to that reported of other rate-reducing agents such as UL-FS49 and ZD7288.Bound drug molecules do not detach from the binding site in the absence of inward current through channels, even if channels are open and the drug is therefore not "trapped" by closed gates.The use-dependence resulting from specific features of I(f) block by ivabradine amplifies its rate-reducing ability at high spontaneous rates and may be useful to clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Department of General Physiology and Biochemistry, Laboratory of Molecular Physiology and Neurobiology, and INFM-Unità Milano Università, 20133 Milano, Italy.

ABSTRACT
"Funny" (f-) channels have a key role in generation of spontaneous activity of pacemaker cells and mediate autonomic control of cardiac rate; f-channels and the related neuronal h-channels are composed of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel subunits. We have investigated the block of f-channels of rabbit cardiac sino-atrial node cells by ivabradine, a novel heart rate-reducing agent. Ivabradine is an open-channel blocker; however, block is exerted preferentially when channels deactivate on depolarization, and is relieved by long hyperpolarizing steps. These features give rise to use-dependent behavior. In this, the action of ivabradine on f-channels is similar to that reported of other rate-reducing agents such as UL-FS49 and ZD7288. However, other features of ivabradine-induced block are peculiar and do not comply with the hypothesis that the voltage-dependence of block is entirely attributable to either the sensitivity of ivabradine-charged molecules to the electrical field in the channel pore, or to differential affinity to different channel states, as has been proposed for UL-FS49 (DiFrancesco, D. 1994. Pflugers Arch. 427:64-70) and ZD7288 (Shin, S.K., B.S. Rotheberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91-101), respectively. Experiments where current flows through channels is modified without changing membrane voltage reveal that the ivabradine block depends on the current driving force, rather than voltage alone, a feature typical of block induced in inwardly rectifying K(+) channels by intracellular cations. Bound drug molecules do not detach from the binding site in the absence of inward current through channels, even if channels are open and the drug is therefore not "trapped" by closed gates. Our data suggest that permeation through f-channel pores occurs according to a multiion, single-file mechanism, and that block/unblock by ivabradine is coupled to ionic flow. The use-dependence resulting from specific features of I(f) block by ivabradine amplifies its rate-reducing ability at high spontaneous rates and may be useful to clinical applications.

Show MeSH
Related in: MedlinePlus