Allosteric interactions between RyR channels justify intracellular Ca2+ release of skeletal muscle in quantitative detail

Figure 1.. Components of a junction between a transverse (T) tubule and an SR terminal cisterna in skeletal muscle. (A) Freeze-dried junctional SR membrane from guinea pig showing RyR1 tetramers in checkerboard formation. (B) Tetrads of CaVs in a freeze-fractured T tubule membrane from toadfish muscle, presented with the same orientation and magnification. (C) Canonical couplon, in which every other RyR is in contact with CaVs (orange elements), thus constituting the “skipping pattern.” The diagram illustrates chirality or handedness, as viewed from outside the cell, in the way RyR tetramers make mutual contact. This orientation, called right-handed, has the inter-tetramer approaches occurring at the tetramers’ edge, to the right-hand side of every corner. Relabeled Fig. 1 of Ríos et al. (2019), which includes images from Block et al. (1988).

Figure 2.. Ca 2+ currents through RyR channels of rabbit skeletal muscle reconstituted in bilayers, exiting a trans compartment with ∼50 mM free [Ca 2+ ]. Republished from Porta et al. (2012). (A and B) The authors interpret that the record in A arises from a set of four channels and those in B from one of five or six channels. Channels can be seen to open or close either individually or together in pairs, in what the authors call “partial coupling.” Records are compared with simulations in Figs. 19 and 20. Republication authorized by Prof. Julio Copello. Fig. 2 is reprinted with permission from American Journal of Physiology.

Figure 3.. Line scan images of Ca 2+ -monitoring fluorescence of fluo-3, elicited by depolarization in patterns shown at top in a myofiber from rat EDL—extensor digitorum longus—muscle (republished from Csernoch et al., 2004). (A and B) In A and B, the events of Ca2+ release were elicited by voltage clamp pulses of very low voltage. (C and D) In C and D, the release system was partially inactivated by a previous depolarization, causing events elicited at higher voltages to be fewer and separate. The traces plot the fluorescence intensity at the sites marked by arrows. The increase over baseline (F0) was intermittent during the pulses and much lower than in spontaneous spark-like events seen in the side pools of the Vaseline-clamp device, where the fiber membrane is destroyed with detergent. Events in side pools are shown in Fig. 1 of the same article. Fig. 3 is reprinted with permission from Journal of Physiology.

Figure 4.. Ca 2+ release flux and its correction. (A) Ca2+ release flux derived from Ca2+ signals of ApIII in a rat EDL myofiber under voltage clamp; flux rises to a peak and decays to a stage that at low voltages is steady and at higher voltages decays slowly. (B) Flux in a speed-optimized clamp setup. (B–F) Mouse interosseus fibers perfused with Fura-2 (B, C, and F) or Fura-FF (D and E). (C) Correction for depletion yields a signal proportional to release membrane permeability. (D) Times to peak permeability from Fura-FF record. (E and F) Peaks of flux and corrected flux or permeability vs. voltage. Partial reproductions of Fig. 3 in Shirokova et al. (1996) (A) and of Fig. 9 in Ursu et al. (2005) (B–F), with labels added. Fig. 4 reprinted with permission from Journal of Physiology.

Figure 5.. Evolution of total released Ca 2+ in a voltage-clamped frog myofiber. (A) Total released Ca2+ vs. time. Ca2+ concentration was measured with Fura-2 and total released was quantified as the increase in total cytoplasmic Ca2+ (Δ[CaT]), mostly bound by EGTA in the experiment. Flux is the rate of change of the records in A (dΔ[CaT]/dt). (B) Semilog plot of flux vs. applied voltage, with a slope corresponding to an e-fold increase every 3.73 mV. Reproduced Fig. 14 in Pape et al. (1995).

Figure 6.. Quantal Ca 2+ release and deterministic inactivation. (A) Superimposed records of flux elicited by the two-pulse patterns shown in a voltage clamped frog myofiber. The presence of a conditioning pulse reduces the peak of the flux elicited by the large test pulse Pref to a value Pcond. The difference Pref-Pcond, called “suppression,” is approximately equal to “decay” of flux (Pc-Sc) elicited by the conditioning pulse. Conditioning does not change the steady value S reached at the end of the test pulse. Reproduced from Pizarro et al. (1997). (B) A set of fluxes elicited by pairs of pulses in rat EDC muscle; a large test pulse follows a conditioning pulse of variable amplitude. (C) The values of decay in the flux elicited by the conditioning pulses of panel B, plotted vs. the suppression induced in the test flux. (D) Replications of the same plot in different individuals. From Szentesi et al. (2000), authorized by Péter Szentesi and László Csernoch. Fig. 6 reprinted with permission from Journal of Physiology.

Figure 7.. Increment detection in Rana. Ca2+ flux in response to the three-step increase in voltage shown. Inset, three patterns of transient response to two steps of stimulus intensity (from Meyer and Stryer, 1990): a, “classical” inactivation, if complete, results in no response to a second step; b, desensitization (also known as adaptation), resulting in a smaller response to the second step; c, the ideal increment detection, with responses proportional to stimulus increment. From Pizarro et al. (1997).

Figure 8.. Calcium transients and release flux activated by a train of pulses. (A–C) Superimposed cytosolic [Ca2+] transients (B) and derived depletion-corrected Ca2+ release flux (C) elicited by the two pulse patterns in A. The nearly complete (91%) suppression of release in the pulses that follow the first one is not altered in spite of the large variation in cytosolic [Ca2+]. Reproduced from Pizarro et al. (1997). Fig. 8 reprinted with permission from Journal of Physiology.

Figure 9.. Gating of independent and allosterically coupled identical channels. (A) The transitions of two channels gating independently. (B) Assumed effects of coupling, in a transition rate theory formulation. The curve in green depicts Gibbs free energy along the transition path of a channel gating independently. In red is the curve modified by coupling to an open channel. In grey is a shifted version of the modified curve, corresponding to a shifted definition of energies, which simplifies the algebra and clarifies the allosteric effects without altering the physics (see note in text). (C) How the two “native” kinetic transition rates, k− and k+, are modified by the interaction with an open channel. (D) Illustrates a requirement for microscopic reversibility, namely K1K4=K2K3, the implications of which are developed in Box 1.

Figure 10.. Channel states and allosteric interactions. (A) State diagram for C channels. O: open; C: closed, I1 and I2: inactivated. V channels only have O and C states. Note irreversibility of O→I1 and I1→C transitions. (B) Illustrates the allosteric energy change in the opening transition of channel 3, when one of its three linked channels (#4) is open and the others (#1 and #5) are closed. Upon opening, all three interactions of #3 change: two CC are lost (with #1 and #5), replaced by two OC, and gains one OO (with #4), replacing one OC, for the net energy change listed. (B) Also illustrates crucial features of the model couplon. The two categories, V and C, are represented in different colors (which one is V is not important here). Note that in large couplons every channel except two at each end interacts with three others, all of which are in the “other” category (every red one with three green ones, and vice versa). In 4-channel couplons every channel interacts just with two others. An N value of 30 (i.e., 30 V and 30 C channels) was used for the simulations of macroscopic or cell-level flux. Simulations for comparison with bilayer data were done with totals of four channels (N = 2)…

Figure 11.. Ca 2+ release flux elicited by constant energy pulses, color-coded in the top diagram. This is the model output to be compared with experimental records of depletion-corrected flux (as in Figs. 4 B, 6 B, or 8 C). Each record is an average of 800 Markov chain realizations. The voltage changes corresponding to each pulse energy are specified in the text. The model parameters used for calculating these and all other averages are always the same, listed in Table 1. Flux, derived from the numbers of V and C channels open via Eq. 7, is given in units of the flux through an open V channel.