Regulation of ryanodine receptors by calsequestrin: effect of high luminal Ca2+ and phosphorylation

Abstract

Calsequestrin, the major calcium sequestering protein in the sarcoplasmic reticulum of muscle, forms a quaternary complex with the ryanodine receptor calcium release channel and the intrinsic membrane proteins triadin and junctin. We have investigated the possibility that calsequestrin is a luminal calcium concentration sensor for the ryanodine receptor. We measured the luminal calcium concentration at which calsequestrin dissociates from the ryanodine receptor and the effect of calsequestrin on the response of the ryanodine receptor to changes in luminal calcium. We provide electrophysiological and biochemical evidence that: 1), luminal calcium concentration of >/=4 mM dissociates calsequestrin from junctional face membrane, whereas in the range of 1-3 mM calsequestrin remains attached; 2), the association with calsequestrin inhibits ryanodine receptor activity, but amplifies its response to changes in luminal calcium concentration; and 3), under physiological calcium conditions (1 mM), phosphorylation of calsequestrin does not alter its ability to inhibit native ryanodine receptor activity when the anchoring proteins triadin and junctin are present. These data suggest that the quaternary complex is intact in vivo, and provides further evidence that calsequestrin is involved in the sarcoplasmic reticulum calcium signaling pathway and has a role as a luminal calcium sensor for the ryanodine receptor.

FIGURE 1

Increasing trans Ca²⁺ from 1 to 5 mM resulted in a biphasic RyR activation. (A–E) Records of 2 s of single channel activity. Single channel opening is upward from zero current (solid line) to maximum open conductance (dashed line). (A) Control, with 1 mM trans Ca²⁺; (B) 30 s after increasing trans Ca²⁺ to 5 mM; (C) 3 min after increasing trans Ca²⁺ to 5 mM; (D) after perfusing the trans chamber with 1 mM Ca²⁺; (E) addition of 16 μg/ml CSQ; (F) average data (n = 5–6) for fractional mean current (I′_F) for conditions shown in panels A–E. (G) Control, with 1 mM trans Ca^2+; (H) after perfusing the trans chamber with 5 mM Ca²⁺, after CSQ dissociation; (I) 5 min after addition of 16 μg/ml CSQ. Average P_o (n = 5) is given to the right of panels G–I. Asterisks (*) indicate average values significantly (p < 0.05, t-test) different from control, and double asterisk (**) and double cross-hatch (##) indicate average values significantly different (p < 0.05, t-test and sign test, respectively) from the previous condition. V_m-E_Cs+ was +40 mV for all records presented in Figs. 1, 2, 4, and 5, except where indicated.

FIGURE 2

Extended exposure to trans Ca²⁺ of 4 mM, and not 2 or 3 mM, resulted in a biphasic RyR activation. (A–E) Records of 2 s of single channel activity. Single channel opening is upward from zero current (solid line) to maximum open conductance (dashed line) for the left panels, and downwards from zero current (solid line) to maximum open conductance (dashed line) for the middle and right panels. (A) Control, with 1 mM trans Ca²⁺; (B) 30 s after increasing trans Ca²⁺ to 4 mM (left panel), 3 mM (middle panel), and 2 mM (right panel); (C) 3 min (middle and right panels) and 9 min (left panel) after increasing trans Ca²⁺ to 4, 3, or 2 mM; (D) after perfusing trans chamber with 1 mM Ca^2+; (E) addition of 16 μg/ml CSQ; (F) average and individual data (n = 4–8 for A–D and n = 1 for E) for fractional mean current (I′_F), at [Ca²⁺]s listed under the graph (left panel) or for conditions shown in panels A–D (middle, right panels). Asterisks (*) indicate average values significantly (p < 0.05, t-test) different from control, and double asterisk (**) and double cross-hatch (##) indicate average values significantly different (p < 0.05, t-test and sign test, respectively) from the previous condition. V_m-E_Cs+ was +40 mV for left panel and −40 mV for middle and right panels.

FIGURE 3

Only [Ca²⁺] ≥4 mM can dissociate CSQ from the solubilized junctional face membrane. (A) Ten percent SDS polyacrylamide gel showing the original junctional face membrane pellet (JFM), insoluble junctional face membrane pellet (P), and solubilized supernatant (S), after the junctional face membrane was exposed to 1, 2, 3, 4, 5, or 10 mM Ca²⁺. CSQ was absent in the solubilized sample (S, lanes 3 and 5) after exposure to 1 or 2 mM Ca²⁺, with the gel profile of the insoluble pellet (P, lanes 2 and 4) being identical to the original junctional face membrane sample (JFM, lane 1). Only trace amounts of CSQ were found in the solubilized sample (S, lane 7) after incubation with 3 mM Ca²⁺, leaving the profiles of the original junctional face membrane and the insoluble pellet (compare lanes 1 and 6) virtually identical. In contrast, increasing amounts of CSQ were dissociated from the original junctional face membrane sample by exposure to 4, 5, and 10 mM Ca²⁺ (S, lanes 9, 11, and 13) with significantly reduced (P, lane 8) or undetectable levels of CSQ observed in the insoluble pellet (P, lanes 10 and 12). (B) Immunoblot of protein products shown in panel A, immunoprobed with VIIID1₂ monoclonal anti-CSQ antibody. Approximate position of the molecular weight markers are indicated to the left of lane 1 in panel A, with CSQ indicated by the arrows. As [Ca²⁺] was kept at 1 mM throughout the CSQ purification procedure, no additional Ca²⁺ was added to the 1-mM sample. The following amount of protein was added to the appropriate lanes; 50 μg original junctional face membrane, 40 μg pellet, and 10 μl solubilized supernatant (which equates to 2.7 μg in lane 9, 3.3 μg in lane 11, and 4.7 μg in lane 13). As a protein concentration cannot be determined for the supernatants obtained after 1, 2, and 3 mM Ca²⁺ extraction, an equivolume (10 μl) of all supernatant fractions was loaded for both the Coomassie stained gel and the immunoblot.

FIGURE 4

RyR responses to changes in luminal [Ca²⁺] between 1 and 5 mM, in the presence and absence of CSQ. Experimental conditions: cis (mM) 250 Cs⁺, 2 ATP (activating), and 1 nM Ca²⁺(subactivating); trans (mM) 250 Cs⁺, 1–5 Ca²⁺. Trans [Ca²⁺] was altered by aliquot additions of 200 mM stock Ca²⁺ ([Ca²⁺] determined by a Ca²⁺ electrode). CSQ was dissociated from RyRs before increasing [Ca²⁺] by exposure to 500 mM Cs⁺, or to high [Ca²⁺] (in the case of the data obtained at 5 mM Ca²⁺). Each data point is the mean relative open probability (relative P_o), which is test P_o (2–5 mM Ca²⁺) relative to control P_o (1 mM), for RyRs in the absence (CSQ(−); ▪) and presence (CSQ(+); ▴) of CSQ. The bars are means ± SE for n = 4–14. Absolute mean P_o values for control activities are listed in the Results section. The inset chart shows relative P_o changes at luminal Ca²⁺ from 1 to 2.0 mM Ca²⁺ in more detail. Asterisks (*) indicate average values significantly different from control (p > 0.05, t-test).

FIGURE 5

Phosphorylation status of CSQ in skeletal muscle; both exogenous phosphorylated and dephosphorylated CSQ inhibit CSQ(−) RyRs. (A) ³¹P-NMR spectrum. The resonances for phosphorylated (P) CSQ, from the original CSQ sample are indicated with arrows in the top trace, whereas residual unbound phosphorous in the dephosphorylated (deP) CSQ, is indicated with an arrow in the bottom trace; (B) Immunoblot of 20 μg P-CSQ and deP-CSQ, immunoprobed with polyclonal antibody anti-phosphothreonine, with the approximate position of molecular weight markers indicated next to the immunoblot; (C and D) changes in CSQ(−) RyR activity after addition of exogenous P-CSQ (C) and deP CSQ (D). In panels C and D, records of 2 s of single channel activity where channel opening is upward from zero current (solid line) to maximum open conductance (dashed line). The top traces show control (Con) activity with 250 mM trans Cs⁺; the middle traces shows activity after increasing trans Cs⁺ to 500 mM with CsMS (Diss); the bottom trace shows addition of 16 μg/ml of P-CSQ (C) or DeP-CSQ (D), after trans perfusion with 250 mM Cs⁺. Average channel P_o values (with means ± SE) are given to the right of traces in panel C (n = 6) and panel D (n = 5). Asterisks (*) indicate average values significantly different from control and double asterisks (**) indicate values different from the previous condition (p < 0.05, t-test).

FIGURE 6

Interactions of triadin and junctin with CSQ are independent of phosphorylation status. Western blot, after 10% SDS-PAGE, of the GST-CSQ coupled to glutathione Sepharose 4B before (lanes 2 and 4) and after (lanes 3 and 5) exposure to solubilized junctional face membrane (JFM; lane 1). Blot was immunoprobed with anti-triadin and anti-junctin. Both phosphorylated (P-CSQ; lanes 2 and 3) and dephosphorylated (deP-CSQ; lanes 4 and 5) CSQ could bind triadin and junctin (lanes 3 and 5) under close to physiological conditions (1 mM Ca²⁺, 150 mM NaCl).