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

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.

Figure 1.

Ivabradine blocks I_f. (A) Structure of ivabradine. (B) Activating/deactivating steps (−100 mV*1.8 s/+5 mV*0.45 s) were applied every 6 s from a holding potential of −35 mV, and a given concentration of ivabradine perfused until full block developed. The time-course of I_f amplitude at −100 mV during block onset and removal is shown for three cells challenged with 0.3 (left), 3 (middle) and 30 μM ivabradine (right). Lower panels show current traces recorded just before and during block development (a to c). Zero current level drawn as a full line. C: dose–response relationship of I_f block by ivabradine from a total of n = 32 cells (mean ± SEM). Each cell was exposed to one drug dose only. Mean data points were fitted to the Hill equation y = 1/(1 + (IC₅₀/x)^h) where x is drug concentration, IC₅₀ the half-block concentration and h the Hill factor (full line: best fitting values in text).

Figure 2.

I_f block by ivabradine and block removal require open channels. (A) Time-course of I_f amplitude at −100 mV during an activation/deactivation protocol (−100/+5 mV, 1/6 Hz) from a holding potential of −35 mV. At the beginning of the perfusion with ivabradine (3 μM), the protocol was interrupted for 100 s while the cell was held at the holding potential. During this time no current reduction was observed. (B) In another cell, the same protocol was applied in the presence of the drug (3 μM) until full block developed. The protocol was interrupted and the cell held at −35 mV for 90 s while simultaneously removing the drug from the perfusing solution. During this time, no reversal of current inhibition occurred. Sample traces shown in the bottom panels were recorded at various times as indicated.

Figure 3.

I_f block by ivabradine depends on the voltage protocol used to activate the current. (A) I_f in control (cont) and after full block by ivabradine (3 μM, asterisks) induced by activation/deactivation protocols at −70 mV (top) and −100 mV (bottom) in two cells. (B) Action of the same drug concentration when applied during steady-state I_f activation at −70 mV (top) or −100 mV (bottom) in two cells.

Figure 4.

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 I_f 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).

Figure 5.

Voltage dependence of steady-state f-channel block by 3 μM ivabradine. Measurements were made by two protocols. At voltages more negative than −40 mV (open circles) I_f was activated by long steps to test potentials and the drug perfused after steady-state activation had been reached; fractional block was measured as the ratio between blocked and control current amplitude. At voltages equal or more positive than −40 mV (filled circles), the membrane was held at the test voltage and a fixed activating voltage step (−100 mV*1.2 s) was applied repetitively (1/6 Hz); the drug was perfused during this protocol and fractional block measured for each test voltage as the ratio between blocked and control current at −100 mV at steady-state. Each point represents the mean ± SEM from 3–5 cells.

Figure 6.

Inward current is required to relieve channel block by ivabradine. I_f block by 3 μM ivabradine was induced by a standard activation/deactivation protocol (−100/+5 mV); sample traces are shown on the left (cont, control; a, 30 s; and b, 120 s after drug perfusion; the latter corresponding to steady-state block). In the continuous presence of the drug, a prolonged (45 s) step to −100 mV was then applied while simultaneously adding 5 mM Cs⁺ (c); Cs⁺ was washed off at the end of the long step and the repetitive pulsing protocol resumed (traces d, 6 s and e, 36 s after Cs⁺ wash-out). Note that no block removal occurred during the prolonged hyperpolarization (compare records b and d in the inset).

Figure 7.

Dependence of the ivabradine-induced I_f block by the current driving force. (A) Sample I_f traces from two cells showing the different degrees of steady-state block induced by 3 μM ivabradine at the same test potential of −30 mV with normal (140 mM, left) and reduced (35 mM, right) external Na⁺ concentration, as measured by activation/deactivation protocols (−100/−30 mV). Fractional block was ∼24% in normal and 54% in reduced Na⁺ concentration. Notice that at −30 mV, as expected, the deactivating I_f tail was inward in normal Na⁺, and outward in reduced Na⁺ conditions. (B) Comparison between mean fractional block curve in normal Tyrode solution (filled circles, as from Fig. 5) and in lowered Na⁺ (open circles). Each point of the curve in low Na⁺ represents the mean ± SEM from 3–6 cells. Vertical dotted lines correspond to the I_f reversal potentials measured from mean fully activated I/V relations from n = 7 cells in the two conditions (E_f = −16.0 mV in normal Tyrode and E_f = −34.4 mV in 35 mM Na⁺ as indicated). Arrows show the intercepts of the block curves with corresponding E_f values.

Figure 8.

Ivabradine causes inward rectification of I_f, which depends on E − E_f. Top: mean fully activated I/V relations measured from n = 7 cells exposed to both normal Tyrode solution (filled circles) and reduced (35 mM) Na⁺ concentration (open circles). I/V relations were measured as described previously (DiFrancesco et al., 1986), by applying pairs of steps, one to fully activate (1 s to −125 mV) and one to fully deactivate I_f (1 s to 15 mV), each followed by a step to the same test voltage, where the amplitude of the different current was measured. Mean ± SEM values are plotted. Linear fitting (straight lines) yielded reversal potentials (E_f) of −16.0 and −34.4 mV for normal and reduced Na⁺ concentration, respectively. Bottom: same curves, multiplied by fractional block values in normal and low Na⁺ solution as deduced from data in Fig. 7 at corresponding voltage and Na⁺ concentration. Strong rectification is apparent in the outward part of both curves, independently of the reversal potential. Lines through points.