Subconductance states in single-channel activity of skeletal muscle ryanodine receptors after removal of FKBP12.
FKBP12 was removed from ryanodine receptors (RyRs) by incubation of rabbit skeletal muscle terminal cisternae membranes with rapamycin. The extent of FKBP12 removal was estimated by immunostaining Western blots of terminal cisternae proteins. Single FKBP12-depleted RyR channels, incorporated into planar lipid bilayers, were modulated by Ca2+, ATP, ryanodine, and ruthenium red in the cis chamber and opened frequently to the normal maximum conductance of approximately 230 pS and to substate levels of approximately 0.25, approximately 0.5, and approximately 0.75 of the maximum conductance. Substate activity was rarely seen in native RyRs. Ryanodine did not after the number of conductance levels in FKBP12-depleted channels, but, at a membrane potential of +40 mV, reduced both the maximum and the substate conductances by approximately 50%. FKBP12-stripped channels were activated by a 10-fold-lower [Ca2+] and inhibited by a 10-fold-higher [Ca2+], than RyRs from control-incubated and native terminal cisternae vesicles. The open probability (Po) of these FKBP12-deficient channels was greater than that of control channels at 0.1 microM and 1 mM cis Ca2+ but no different at 10 microM cis Ca2+, where channels showed maximal Ca2+ activation. The approximately 0.25 substate was less sensitive than the maximum conductance to inhibition by Ca2+ and was the dominant level in channels inhibited by 1 mM cis Ca2+. The results show that FKBP12 coordinates the gating of channel activity in control and ryanodine-modified RyRs.
Ahern
G P
GP
Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, Australia.
Junankar
P R
PR
Dulhunty
A F
AF
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D004553:Electric Conductivity; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D015640:Ion Channel Gating; D008051:Lipid Bilayers; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000187:drug effects / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011090:Polyenes / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D012430:Ruthenium Red / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000502:physiology; D020123:Sirolimus; D022021:Tacrolimus Binding Proteins
D000906:Antibodies; D015220:Calcium Channels; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D007166:Immunosuppressive Agents; D008051:Lipid Bilayers; D009124:Muscle Proteins; D010446:Peptide Fragments; D011090:Polyenes; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; D012433:Ryanodine; D000255:Adenosine Triphosphate; D022021:Tacrolimus Binding Proteins; D002118:Calcium; D020123:Sirolimus