Voltage-dependent modulation of cardiac ryanodine receptors (RyR2) by protamine

Abstract

It has been reported that protamine (>10 microg/ml) blocks single skeletal RyR1 channels and inhibits RyR1-mediated Ca2+ release from sarcoplasmic reticulum microsomes. We extended these studies to cardiac RyR2 reconstituted into planar lipid bilayers. We found that protamine (0.02-20 microg/ml) added to the cytosolic surface of fully activated RyR2 affected channel activity in a voltage-dependent manner. At membrane voltage (V(m); SR lumen-cytosol) = 0 mV, protamine induced conductance transitions to several intermediate states (substates) as well as full block of RyR2. At V(m)>10 mV, the substate with the highest level of conductance was predominant. Increasing V(m) from 0 to +80 mV, decreased the number of transitions and residence of the channel in this substate. The drop in current amplitude (full opening to substate) had the same magnitude at 0 and +80 mV despite the approximately 3-fold increase in amplitude of the full opening. This is more similar to rectification of channel conductance induced by other polycations than to the action of selective conductance modifiers (ryanoids, imperatoxin). A distinctive effect of protamine (which might be shared with polylysines and histones but not with non-peptidic polycations) is the activation of RyR2 in the presence of nanomolar cytosolic Ca2+ and millimolar Mg2+ levels. Our results suggest that RyRs would be subject to dual modulation (activation and block) by polycationic domains of neighboring proteins via electrostatic interactions. Understanding these interactions could be important as such anomalies may be associated with the increased RyR2-mediated Ca2+ leak observed in cardiac diseases.

Figure 1. Effect of protamine on fully activated RyR2.

Single channel recordings of a RyR2 channel fully activated by the combined action of cytosolic Ca²⁺ (10 µM) and caffeine (5 mM). Lumenal (trans) Ca²⁺ (50 mM) was the current carrier. All recordings were performed at holding voltage (V_m) = 0 mV. Channel openings are observed as positives deflections of the current (o = full open; b = baseline). The frequency-current amplitude histograms obtained from 4-min single-channel recordings are shown next to each trace. Representative traces recorded before (top trace) and after addition of increasing concentrations of protamine (0.02, 0.2, 1, 2 and 20 µg/ml). Current levels of protamine-induced substates are indicated by dashed lines. Subsequent addition of 250 µg/ml heparin reversed the effect of protamine (bottom trace). 4 min-recordings were performed in all the above-mentioned conditions (n = 6 experiments).

Figure 2. Effect of voltage changes on protamine-induced substates RyR2.

Single-channel recordings of a RyR2 channel activated with Ca²⁺/caffeine (10 µM/5 mM, respectively). Openings are shown as upward deflections (o = full open; b = baseline). The frequency-current amplitude histograms obtained from 4-min single-channel recordings are shown next to each trace. Representative traces recorded at +30, +40, +50, +60 and +70 mV (trans - cis) are shown under control conditions (A) and after addition of 1 µg/ml protamine (B). p represents openings to the protamine-induced substate. (C) Fraction of time spent at the protamine-induced substate (P_Substate) as a function of holding voltage (V_m) ranging from +30 to +70 mV. Values are averages±SEM of P_Substate calculated from n = 5 recordings. Lines represent fits of the Woodhull equation to experimental data (see Results). (D) The drop in current amplitude from the full opening to the protamine-induced substate (ΔI) and the ratio: ΔI/I_{full opening} were calculated for 10 independent experiments and plotted as a function of holding voltage (open circles and filled circles, respectively). Averages ± SEM of these values are shown as a function of V_m.

Figure 3. Effect of protamine compared to other conductance-modifiers.

Single-channel recordings of fully active RyR2 channels (10 µM cytosolic Ca²⁺/5 mM caffeine) in the presence of 1 µg/ml protamine, 10 µM ryanodol or 50 nM imperatoxin A (A, B and C, respectively). Channel activity was recorded at V_m = 0 mV (top traces) and +40 mV (bottom traces). The current levels for the baseline, full open state and substates induced by protamine, ryanodol and IpTx_A are indicated (b, o, p, r and i, respectively). (D) Current amplitude as a function of voltage for the full open state (n = 28) and for substates induced by protamine (n = 10), ryanodol (n = 13) and IpTx_A (n = 5). Slope conductances were, in pS, 129±1, 123±3, 56±2 and 43±3, respectively.

Figure 4. Current voltage (I-V) curves for the full open state and for the protamine-induced substate.

Current amplitude as a function of voltage was plotted based on single-channel recordings of fully active RyR2 (10 µM cytosolic Ca²⁺/5 mM caffeine) performed in the presence of cytosolic Cs⁺ and 0.2 µg/ml protamine. Values are averages ± SEM of 5 experiments.

Figure 5. Effect of protamine in combination with imperatoxin A (IpTx_A).

RyR2 channels fully activated by the combined action of cytosolic Ca²⁺ (10 µM) and caffeine (5 mM) were used as control (A). Subsequently, 50 nM IpTx_A (B) or 1 µg/ml protamine (C) were added. To test the combined effect of 1 µg/ml protamine + 50 nM IpTx_A two sets of experiments were performed: one adding protamine first and then IpTx_A and the other, switching the order in which these agents were added. The results were the same in both sets of experiments. A representative recording is displayed in panel (D). All traces shown were performed at V_m = +20 mV. Current levels for the baseline (b), full open state (o), protamine-induced substate (p) are indicated. A small substate (s) is indicated. In panels (A) and (B) the amplitude of this substate is ∼30% of the full open state and in panels (C) and (D) it is ∼30% the amplitude of the protamine-induced substate. (E) Probability of substates at the s level (P_S) as a function of holding voltage of RyR2 channels under control conditions (open triangles), exposed to 50 nM IpTx_A (open circles), 1 µg/ml protamine (filled triangles) and 50 nM IpTx_A +1 µg/ml protamine (filled circles). Values are means±SEM (* P<0.05; n = 10 experiments).

Figure 6. Effect of protamine in combination with ryanodol.

Continuous trace of a RyR2 in the presence of 10 µM ryanodol before (A) and after subsequent addition of 1 µg/ml protamine (B) and 250 µg/ml heparin (C). Recordings were performed at V_m = +20 mV (b = baseline; o = full open state; r = ryanodol-induced substate; p = protamine-induced substate; p+r = ryanodol-induced substate on the protamine-modified RyR2). (D) Probability of ryanodol-induced substates (P_Ryanodol) as a function of holding voltage of RyR2 channels exposed to 10 µM ryanodol (open circles); 10 µM ryanodol+1 µg/ml protamine (filled circles) and 10 µM ryanodol+1 µg/ml protamine+250 µg/ml heparin (open triangles). Under these three conditions, the dwell times of the ryanodol-induced events were (in seconds): 3.7±0.7; 11.3±2.5 and 3.4±0.5 respectively. Values are means±SEM (* P<0.05; n = 7 experiments).

Figure 7. Effect of protamine at low cytosolic Ca²⁺.

Single channel recordings of partially active RyR2 in the presence of 100 nM cytosolic Ca²⁺ before (A) and after subsequent addition of 1 µg/ml protamine (B) and 250 µg/ml heparin (C). The frequency-current amplitude histograms obtained from 4-min single-channel recordings are shown next to each trace. Channel activity was recorded at V_m = +20 mV (top traces) and +40 mV (bottom traces). (D) Averages of open probabilities calculated for single RyR2 at V_m = +20 mV (black columns) and +40 mV (shaded columns). Here, the open probability includes the probability of the full opening plus the probability of protamine-induced substates. Values are shown as means±SEM (* P<0.05 versus control; # P<0.05 versus protamine at +20 mV; n = 5 experiments).

Figure 8. Sequence of events for protamine-induced activation of RyR2.

(A) Continuous trace of a RyR2 in the presence of 25 nM cytosolic Ca²⁺ plus 1 µg/ml protamine recorded at V_m = +40 mV (b = baseline; o = full opening; p = protamine-induced substate). The beginning and end of each opening are indicated by grey and black arrows, respectively. (B) Diagram of states of RyR2 exposed to 1 µg/ml protamine with 25 nM cytosolic Ca²⁺ (4 min recording at V_m = +40 mV). The following states were considered: full open, protamine substate, closed. State probabilities (P) are given in parenthesis. Arrows represent observed unidirectional transitions from one state to another. Adjacent to each arrow is the respective absolute number of transitions.