Characterization of ryanodine receptor type 1 single channel activity using "on-nucleus" patch clamp

Abstract

In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca(2+) release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca(2+)] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ∼750pS or 450pS in symmetrical 250mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ∼40% of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca(2+), and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation.

Keywords: Inositol 1,4,5-trisphosphate receptor; Ryanodine receptor; Single channel measurement.

Figure 1. RyR1 and FKBP expression in RyR1 stable Flp-In cells

A) Representative western blot analysis of 10 μg each of HEK-RyR1 and control Flp-In 293 cell lysates probed with anti-RyR antibody. A sarcoplasmic reticulum (SR) preparation (5 μg) from mouse skeletal muscle was used as a positive control. B) Representative western blot analysis of 10 μg each of HEK-RyR1 and control Flp-In 293 cell lysates probed with anti-FKBP (1:100) antibody. A tibialis anterior (TA) mouse muscle homogenate preparation (10 μg) was used as a positive control for FKBP12. C) Control Flp-In 293 cells (Panel I) and HEK-RyR1 cells (Panels II and III) showing Hoechst 34580 nuclear staining alone (left), RyR1 immunostaining alone (middle) and overlay (right).

Figure 2. RyR1 Ca²⁺ release channel function in stable HEK-RyR1 cells

(A) Representative indo-1 ratio traces obtained from HEK-RyR1 cells (red) and control (black) Flp-In 293 cells during application of 500 μM 4-CMC. (B) Average (± SE) 4-CMC concentration-response curves for RyR1 stably expressing (red) and control (black) Flp-In 293 cells. (C) Representative indo-1 ratio traces obtained from HEK-RyR1 cells during application of 10 mM caffeine in the absence (black) and presence (red) of pretreatment with 100 μM ryanodine. (D) Average (± SE) response of HEK-RyR1 cells to 10 mM caffeine in the absence (black) and presence (red) of pretreatment with 100 μM ryanodine. *p < 0.05.

Figure 3. Uncaging [Ca²⁺] in the patch pipette triggers single channel activity

The cartoon illustrates the voltage clamp and ionic conditions. Patch pipette contained (in mM): 250 KCl, 25 HEPES, 10 NP-EGTA, 3 CaCl₂, 0.5 Na-ATP. The holding potential was +40 mV. Free [Ca²⁺] prior to UV flash was <100 nM. Upon UV photolysis, free [Ca²⁺] increased to >10 μM. (A) Representative current traces of this and four other experiments displayed no channel activity prior to UV flash. Upon photolysis, following a short latency, channel activity increased markedly. Openings (O) from the closed state (C) are denoted by upward deflections. (B) Magnifying a 300 ms section of the trace allows better visualization of discrete openings and closings.

Figure 4. Increasing [Ca²⁺] augments open probability through enhancing transition from a closed state

Representative current traces at +40 mV are shown for experiments with pipette solutions containing either 1 μM (A) or 100 μM (B) Ca²⁺. (C) Increasing pipette [Ca²⁺] from 1 μM (black bar) to 100 μM (red bar) increased average channel open probability from 0.04 to 0.36. (D) Mean (± SE) channel open (left) and closed (right) times in the presence of either 1 μM (black bars) or 100 μM (red bars) pipette [Ca²⁺]. All points histograms for open (E, F) and closed times (G, H) were constructed for 1 μM (E, G) and 100 μM (F, H) [Ca²⁺] and fitted with single exponential functions. Time constants for open channel dwell times were unaltered, but τ_closed was 10 times longer in 1 μM Ca²⁺. Thus, higher [Ca²⁺] destabilized the closed state, thereby permitting faster transitions into the open state. *p < 0.05

Figure 5. Ion selectivity favors potassium over cesium

Representative current traces at the voltages listed are shown with symmetrical K⁺ (A) and Cs⁺ (B) as the predominate charge carrying ions. (C) Average (± SE) current-voltage relationships were constructed for both KCl and CsCl experiments. Traces were obtained at test potentials between −100 mV and 100 mV in 10 mV increments. Data were fitted with linear functions and conductances were calculated from the slopes as described in Methods. Reversal potentials were unaltered. However, a profound decrease in the slope conductance from K⁺ (745 ± 18 pS) to Cs⁺ (454 ± 17 pS) as charge carrier was evident. Traces are representative examples of at least 4 other patches.

Figure 6. Caffeine shifts the Ca²⁺ dependence by promoting a transition out of a closed state

(A) Representative current traces are shown before and after addition of 10 mM caffeine. Caffeine increased channel open probability after a latency of several seconds. (B) Average (± SE) open probability histogram, binned in 500 ms increments, shows enhanced channel activity 12 seconds following the addition of 10 mM caffeine. (C) Bar charts constructed of mean (± SE) open (left) and closed (right) dwell times before and after addition of 10 mM caffeine. Only closed times were affected by caffeine. (D) Open (left) and closed (right) time histograms before and after the addition of 10 mM caffeine. Each distribution was well fitted with a single exponential. Time constants are inset into each graph. Open time constants were unaltered, while the closed time constant was reduced, in the presence of caffeine. Like Ca²⁺, caffeine destabilizes the closed state, and thereby facilitates transitions into the open state. Traces are representative examples of at least 4 other patches.

Figure 7. Ryanodine drives the channel into a long-lived sub-conductance state

Representative current traces are shown for recording in the presence of 100 μM Ca²⁺ immediately before and following perfusion of 10 μM ryanodine denoted by the arrow. Following ryanodine addition, the channel entered into a long-lived, sub-conducting state that did not transition back to the normal conductance state for several seconds, Traces are representative examples of at least 4 other patches.

Figure 8. Effects of potential RyR modulators on open probability

Experiments were performed wit each compound listed in the presence of 1 μM Ca²⁺, and separately in the presence of 100 μM Ca²⁺ to demonstrate maximal achievable P_o. Mean (± SE) open probabilities were calculated for each experiment. A bar chart was constructed for the grouped data (n ≥ 4 for each condition) and plotted for each condition. Only caffeine and 100 μM Ca²⁺ had a significant effect on channel open probability in the presence of 1 μM Ca²⁺.*p < 0.05.