Activation and deactivation of sarcoplasmic reticulum calcium release channels: Molecular dissection of mechanisms via novel semi-synthetic ryanoids

Keshore R. Bidasee, Henry R. Besch, Koert Gerzon, Rod A. Humerickhouse

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

The plant alkaloids ryanodine and dehydroryanodine are high affinity, biphasic modulators of the intracellularly located, calcium-regulated calcium release channels of a variety of cell types. To date, little is certain about the molecular basis of the interactions that prompt low concentrations of ryanodine (nanomolar to low micromolar) to activate (open) the channels and higher concentrations to deactivate (functionally close) the sarcoplasmic reticulum calcium release channel. In the present study, we approached this question using novel, semi-synthetic C10-Oeq ester derivatives of ryanodine and dehydroryanodine as molecular probes of the ryanodine binding sites on the calcium release channel. Binding affinities of these C10-Oeq ester derivatives of ryanodine and dehydroryanodine with acidic, basic and neutral side chains (Kd values> 53.9 nM, Kd values 0.3-0.7 nM and Kd values 1.3-20.4 nM, compared with 2.3 and 2.8 nM for ryanodine and dehydroryanodine, respectively) were evaluated for their ability to modulate, the patency of the sarcoplasmic reticulum calcium release channel. With the exception of only two derivatives tested to date, all the semi-synthetic C10-Oeq esters selectively activate the Ca2+ release channel. That is, they produce no functional closure of the sarcoplasmic reticulum calcium release channels at the highest concentration, that could be tested. Half-maximal concentrations for activation (EC50 act, values) ranged from 0.87-4.2, μM, compared with an EC50 act of 1.3 μM for ryanodine. Using a low concentration (0.5 nM) of a high specific activity, radioiodinated derivative of ryanodine, C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine (1400 Ci/mmol) as the radioligand in displacement binding affinity assays, two distinct, sequential ryanodine binding isotherms were demonstrated within the normal 0-300 nM ryanodine sigmoidal displacement curve. A high affinity site had an IC50 of 0.5 nM (Kd=0.26±0.02 nM). Above this concentration, an apparent plateau occurred between 3 and 6 nM ryanodine, and at higher concentrations a lower affinity site, was revealed that demonstrated an IC50 of about 25 nM (Kd=11.7±1.2, nM). Scatchard analysis from direct binding of C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine to junctional sarcoplasmic reticulum vesicles also suggests the presence of more than one class of binding sites within the nanomolar concentration range. The high affinity site, demonstrated a Bmax of 3 pmol/mg protein. We were unable to saturate the lower affinity binding sites with this ligand. To evaluate the functional effects occurring among sarcoplasmic reticulum calcium release channel monomers as a consequence of ryanodine's binding, we utilized a photo-activatable derivative of ryanodine, C10-Oeq N-(4-azido salicyloyl) glycyl ryanodine that demonstrates channel modulating characteristics similar to ryanodine. Covalently labeling the sarcoplasmic reticulum calcium-release channels with this ligand, followed by measurements of rates of calcium efflux and SDS-PAGE of the labeled protein, revealed that deactivation of the sarcoplasmic reticulum calcium release channels of skeletal muscle by this ryanoid occurred at concentrations which apparently produce virtually irreversibly, interactions between receptor monomers. This 'polymerization' was indicated by the progressive appearance of two higher molecular weight protein bands on SDS-PAGE concomitant with progressive decreases in the ryanodine receptor monomer band that runs at an apparent molecular mass of 365 kDa. In summary, we have prepared an utilized, novel C10-Oeq ester derivatives of ryanodine and dehydroryanodine in studies aimed at better understanding the molecular basis for the complex biphasic actions of ryanodine on the sarcoplasmic reticulum calcium release channels from rabbit skeletal muscle cells. The described studies presage correlations that may be useful in furthering our understanding of structure-function relationship among ryanoids.

Original languageEnglish (US)
Pages (from-to)145-160
Number of pages16
JournalMolecular and cellular biochemistry
Volume149-150
Issue number1
DOIs
StatePublished - Aug 1 1995

Fingerprint

Dissection
Ryanodine
Sarcoplasmic Reticulum
Calcium Channels
Chemical activation
Calcium
Derivatives
Esters
Monomers
Binding Sites
Inhibitory Concentration 50
Muscle
Polyacrylamide Gel Electrophoresis
Skeletal Muscle
Ligands
Molecular Probes
Ryanodine Receptor Calcium Release Channel
Proteins

Keywords

  • Ca release channels
  • interactions of novel C-O ester ryanoids with SR-CRC
  • ryanodine receptors
  • sarcoplasmic reticulum

ASJC Scopus subject areas

  • Molecular Biology
  • Clinical Biochemistry
  • Cell Biology

Cite this

Activation and deactivation of sarcoplasmic reticulum calcium release channels : Molecular dissection of mechanisms via novel semi-synthetic ryanoids. / Bidasee, Keshore R.; Besch, Henry R.; Gerzon, Koert; Humerickhouse, Rod A.

In: Molecular and cellular biochemistry, Vol. 149-150, No. 1, 01.08.1995, p. 145-160.

Research output: Contribution to journalArticle

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abstract = "The plant alkaloids ryanodine and dehydroryanodine are high affinity, biphasic modulators of the intracellularly located, calcium-regulated calcium release channels of a variety of cell types. To date, little is certain about the molecular basis of the interactions that prompt low concentrations of ryanodine (nanomolar to low micromolar) to activate (open) the channels and higher concentrations to deactivate (functionally close) the sarcoplasmic reticulum calcium release channel. In the present study, we approached this question using novel, semi-synthetic C10-Oeq ester derivatives of ryanodine and dehydroryanodine as molecular probes of the ryanodine binding sites on the calcium release channel. Binding affinities of these C10-Oeq ester derivatives of ryanodine and dehydroryanodine with acidic, basic and neutral side chains (Kd values> 53.9 nM, Kd values 0.3-0.7 nM and Kd values 1.3-20.4 nM, compared with 2.3 and 2.8 nM for ryanodine and dehydroryanodine, respectively) were evaluated for their ability to modulate, the patency of the sarcoplasmic reticulum calcium release channel. With the exception of only two derivatives tested to date, all the semi-synthetic C10-Oeq esters selectively activate the Ca2+ release channel. That is, they produce no functional closure of the sarcoplasmic reticulum calcium release channels at the highest concentration, that could be tested. Half-maximal concentrations for activation (EC50 act, values) ranged from 0.87-4.2, μM, compared with an EC50 act of 1.3 μM for ryanodine. Using a low concentration (0.5 nM) of a high specific activity, radioiodinated derivative of ryanodine, C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine (1400 Ci/mmol) as the radioligand in displacement binding affinity assays, two distinct, sequential ryanodine binding isotherms were demonstrated within the normal 0-300 nM ryanodine sigmoidal displacement curve. A high affinity site had an IC50 of 0.5 nM (Kd=0.26±0.02 nM). Above this concentration, an apparent plateau occurred between 3 and 6 nM ryanodine, and at higher concentrations a lower affinity site, was revealed that demonstrated an IC50 of about 25 nM (Kd=11.7±1.2, nM). Scatchard analysis from direct binding of C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine to junctional sarcoplasmic reticulum vesicles also suggests the presence of more than one class of binding sites within the nanomolar concentration range. The high affinity site, demonstrated a Bmax of 3 pmol/mg protein. We were unable to saturate the lower affinity binding sites with this ligand. To evaluate the functional effects occurring among sarcoplasmic reticulum calcium release channel monomers as a consequence of ryanodine's binding, we utilized a photo-activatable derivative of ryanodine, C10-Oeq N-(4-azido salicyloyl) glycyl ryanodine that demonstrates channel modulating characteristics similar to ryanodine. Covalently labeling the sarcoplasmic reticulum calcium-release channels with this ligand, followed by measurements of rates of calcium efflux and SDS-PAGE of the labeled protein, revealed that deactivation of the sarcoplasmic reticulum calcium release channels of skeletal muscle by this ryanoid occurred at concentrations which apparently produce virtually irreversibly, interactions between receptor monomers. This 'polymerization' was indicated by the progressive appearance of two higher molecular weight protein bands on SDS-PAGE concomitant with progressive decreases in the ryanodine receptor monomer band that runs at an apparent molecular mass of 365 kDa. In summary, we have prepared an utilized, novel C10-Oeq ester derivatives of ryanodine and dehydroryanodine in studies aimed at better understanding the molecular basis for the complex biphasic actions of ryanodine on the sarcoplasmic reticulum calcium release channels from rabbit skeletal muscle cells. The described studies presage correlations that may be useful in furthering our understanding of structure-function relationship among ryanoids.",
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T2 - Molecular dissection of mechanisms via novel semi-synthetic ryanoids

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N2 - The plant alkaloids ryanodine and dehydroryanodine are high affinity, biphasic modulators of the intracellularly located, calcium-regulated calcium release channels of a variety of cell types. To date, little is certain about the molecular basis of the interactions that prompt low concentrations of ryanodine (nanomolar to low micromolar) to activate (open) the channels and higher concentrations to deactivate (functionally close) the sarcoplasmic reticulum calcium release channel. In the present study, we approached this question using novel, semi-synthetic C10-Oeq ester derivatives of ryanodine and dehydroryanodine as molecular probes of the ryanodine binding sites on the calcium release channel. Binding affinities of these C10-Oeq ester derivatives of ryanodine and dehydroryanodine with acidic, basic and neutral side chains (Kd values> 53.9 nM, Kd values 0.3-0.7 nM and Kd values 1.3-20.4 nM, compared with 2.3 and 2.8 nM for ryanodine and dehydroryanodine, respectively) were evaluated for their ability to modulate, the patency of the sarcoplasmic reticulum calcium release channel. With the exception of only two derivatives tested to date, all the semi-synthetic C10-Oeq esters selectively activate the Ca2+ release channel. That is, they produce no functional closure of the sarcoplasmic reticulum calcium release channels at the highest concentration, that could be tested. Half-maximal concentrations for activation (EC50 act, values) ranged from 0.87-4.2, μM, compared with an EC50 act of 1.3 μM for ryanodine. Using a low concentration (0.5 nM) of a high specific activity, radioiodinated derivative of ryanodine, C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine (1400 Ci/mmol) as the radioligand in displacement binding affinity assays, two distinct, sequential ryanodine binding isotherms were demonstrated within the normal 0-300 nM ryanodine sigmoidal displacement curve. A high affinity site had an IC50 of 0.5 nM (Kd=0.26±0.02 nM). Above this concentration, an apparent plateau occurred between 3 and 6 nM ryanodine, and at higher concentrations a lower affinity site, was revealed that demonstrated an IC50 of about 25 nM (Kd=11.7±1.2, nM). Scatchard analysis from direct binding of C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine to junctional sarcoplasmic reticulum vesicles also suggests the presence of more than one class of binding sites within the nanomolar concentration range. The high affinity site, demonstrated a Bmax of 3 pmol/mg protein. We were unable to saturate the lower affinity binding sites with this ligand. To evaluate the functional effects occurring among sarcoplasmic reticulum calcium release channel monomers as a consequence of ryanodine's binding, we utilized a photo-activatable derivative of ryanodine, C10-Oeq N-(4-azido salicyloyl) glycyl ryanodine that demonstrates channel modulating characteristics similar to ryanodine. Covalently labeling the sarcoplasmic reticulum calcium-release channels with this ligand, followed by measurements of rates of calcium efflux and SDS-PAGE of the labeled protein, revealed that deactivation of the sarcoplasmic reticulum calcium release channels of skeletal muscle by this ryanoid occurred at concentrations which apparently produce virtually irreversibly, interactions between receptor monomers. This 'polymerization' was indicated by the progressive appearance of two higher molecular weight protein bands on SDS-PAGE concomitant with progressive decreases in the ryanodine receptor monomer band that runs at an apparent molecular mass of 365 kDa. In summary, we have prepared an utilized, novel C10-Oeq ester derivatives of ryanodine and dehydroryanodine in studies aimed at better understanding the molecular basis for the complex biphasic actions of ryanodine on the sarcoplasmic reticulum calcium release channels from rabbit skeletal muscle cells. The described studies presage correlations that may be useful in furthering our understanding of structure-function relationship among ryanoids.

AB - The plant alkaloids ryanodine and dehydroryanodine are high affinity, biphasic modulators of the intracellularly located, calcium-regulated calcium release channels of a variety of cell types. To date, little is certain about the molecular basis of the interactions that prompt low concentrations of ryanodine (nanomolar to low micromolar) to activate (open) the channels and higher concentrations to deactivate (functionally close) the sarcoplasmic reticulum calcium release channel. In the present study, we approached this question using novel, semi-synthetic C10-Oeq ester derivatives of ryanodine and dehydroryanodine as molecular probes of the ryanodine binding sites on the calcium release channel. Binding affinities of these C10-Oeq ester derivatives of ryanodine and dehydroryanodine with acidic, basic and neutral side chains (Kd values> 53.9 nM, Kd values 0.3-0.7 nM and Kd values 1.3-20.4 nM, compared with 2.3 and 2.8 nM for ryanodine and dehydroryanodine, respectively) were evaluated for their ability to modulate, the patency of the sarcoplasmic reticulum calcium release channel. With the exception of only two derivatives tested to date, all the semi-synthetic C10-Oeq esters selectively activate the Ca2+ release channel. That is, they produce no functional closure of the sarcoplasmic reticulum calcium release channels at the highest concentration, that could be tested. Half-maximal concentrations for activation (EC50 act, values) ranged from 0.87-4.2, μM, compared with an EC50 act of 1.3 μM for ryanodine. Using a low concentration (0.5 nM) of a high specific activity, radioiodinated derivative of ryanodine, C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine (1400 Ci/mmol) as the radioligand in displacement binding affinity assays, two distinct, sequential ryanodine binding isotherms were demonstrated within the normal 0-300 nM ryanodine sigmoidal displacement curve. A high affinity site had an IC50 of 0.5 nM (Kd=0.26±0.02 nM). Above this concentration, an apparent plateau occurred between 3 and 6 nM ryanodine, and at higher concentrations a lower affinity site, was revealed that demonstrated an IC50 of about 25 nM (Kd=11.7±1.2, nM). Scatchard analysis from direct binding of C10-Oeq N-(4-azido-5-125iodo salicyloyl) glycyl ryanodine to junctional sarcoplasmic reticulum vesicles also suggests the presence of more than one class of binding sites within the nanomolar concentration range. The high affinity site, demonstrated a Bmax of 3 pmol/mg protein. We were unable to saturate the lower affinity binding sites with this ligand. To evaluate the functional effects occurring among sarcoplasmic reticulum calcium release channel monomers as a consequence of ryanodine's binding, we utilized a photo-activatable derivative of ryanodine, C10-Oeq N-(4-azido salicyloyl) glycyl ryanodine that demonstrates channel modulating characteristics similar to ryanodine. Covalently labeling the sarcoplasmic reticulum calcium-release channels with this ligand, followed by measurements of rates of calcium efflux and SDS-PAGE of the labeled protein, revealed that deactivation of the sarcoplasmic reticulum calcium release channels of skeletal muscle by this ryanoid occurred at concentrations which apparently produce virtually irreversibly, interactions between receptor monomers. This 'polymerization' was indicated by the progressive appearance of two higher molecular weight protein bands on SDS-PAGE concomitant with progressive decreases in the ryanodine receptor monomer band that runs at an apparent molecular mass of 365 kDa. In summary, we have prepared an utilized, novel C10-Oeq ester derivatives of ryanodine and dehydroryanodine in studies aimed at better understanding the molecular basis for the complex biphasic actions of ryanodine on the sarcoplasmic reticulum calcium release channels from rabbit skeletal muscle cells. The described studies presage correlations that may be useful in furthering our understanding of structure-function relationship among ryanoids.

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KW - interactions of novel C-O ester ryanoids with SR-CRC

KW - ryanodine receptors

KW - sarcoplasmic reticulum

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