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Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (alpha1S) expressing two complementary protein fragments.

Ahern CA, Vallejo P, Mortenson L, Coronado R - BMC Physiol. (2001)

Bottom Line: Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca2+ current.In these cases, function is recovered by expression of complementary protein fragments from the same cDNA.DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca2+ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706, USA. cahern@physiology.wisc.edu

ABSTRACT

Background: The L-type Ca2+ channel formed by the dihydropyridine receptor (DHPR) of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1). This channel-to-channel coupling is essential for Ca2+ signaling but poorly understood. We characterized a single-base frame-shift mutant of alpha1S, the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC) coupling by virtue of expressing two complementary hemi-Ca2+ channel fragments.

Results: Functional analysis of cDNA transfected dysgenic myotubes lacking alpha1S were carried out using voltage-clamp, confocal Ca2+ indicator fluoresence, epitope immunofluorescence and immunoblots of expressed proteins. The frame-shift mutant (fs-alpha1S) expressed the N-terminal half of alpha1S (M1 to L670) and the C-terminal half starting at M701 separately. The C-terminal fragment was generated by an unexpected restart of translation of the fs-alpha1S message at M701 and was eliminated by a M701I mutation. Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca2+ current.

Discussion: A premature stop codon in the II-III loop may not necessarily cause a loss of DHPR function due to a restart of translation within the II-III loop, presumably by a mechanism involving leaky ribosomal scanning. In these cases, function is recovered by expression of complementary protein fragments from the same cDNA. DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca2+ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel.

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EC coupling generated by two complementary fragments of α1S. Line scans (horizontal dimension is 2.05 seconds) of fluo-4 fluorescence show Ca2+ transients in response to the indicated 50-ms depolarization from a holding potential of -40 mV. Trace of integrated fluorescence in ΔF/Fo units is shown for each line scan. Each set of depolarizations is from a separate dysgenic myotube expressing the α1S construct(s) indicated at the top of each column. A 16-color calibration bar in ΔF/Fo units is included in Fig. 3 for visual reference.
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Figure 5: EC coupling generated by two complementary fragments of α1S. Line scans (horizontal dimension is 2.05 seconds) of fluo-4 fluorescence show Ca2+ transients in response to the indicated 50-ms depolarization from a holding potential of -40 mV. Trace of integrated fluorescence in ΔF/Fo units is shown for each line scan. Each set of depolarizations is from a separate dysgenic myotube expressing the α1S construct(s) indicated at the top of each column. A 16-color calibration bar in ΔF/Fo units is included in Fig. 3 for visual reference.

Mentions: The EC coupling recovered by fs-α1S could be due either to the activity of the N-terminal half of α1S alone or to protein-protein complementation between the N-terminal half and a fragment expressing the C-terminal half of α1S. The C-terminal half of α1S could have been translated by the fs-α1S expression vector if the ATG codon (Met701), which is downstream from the TGA termination codon and is in-frame with the wild-type message (Fig 1B) served as open reading frame for translation of the second half of the wt message. Although this would be unusual, the fact that the codon for Met701 is only 25 bases downstream from the termination codon could have substantially increased the possibility of a re-start of the translation of the second half of the message at Met701. This phenomenon has been described in eukaryotic cells and in viral-infected mammalian cells and is known as translation by leaky ribosomal scanning [32,33]. To test this explanation, the presumptive restart condon, Met701, was mutated to Ile701 in the fs-α1S template. If fs-α1S recovered EC coupling by virtue of expressing a single protein fragment, then fs-α1SM701I should also recover EC coupling since the mutation was introduced downstream from the stop codon. Fig. 5 shows that this was not the case. Fs-α1SM701I did not recover Ca2+ transients in 9 of 9 tested cells, consistent with leaky ribosomal scanning. As a positive control, we coexpressed fs-α1SM701I and the C-terminus half of α1S, namely α1SΔ1–700, cloned into a separate pSG5 vector. The results in Fig. 5 indicated that α1SΔ1–700 alone was inactive. However, when myotubes were cotransfected with fs-α1SM701I and α1SΔ1–700, each in a separate pSG5 vector, there was a robust recovery of Ca2+ transients in 5 of 5 cells. Fig. 6A shows fluorescence vs. voltage relationships for the fs-α1SM701I mutant and for this mutant coexpressed with α1SΔ1–700. The combined expression of the two complementary fragments of α1S resulted in a robust recovery of EC coupling with sigmoidal Ca2+ release vs. voltage characteristics. A summary of the maximum fluorescence during the Ca2+ transient in response to a depolarization to +90 mV is shown in Fig. 6B. The magnitude of the Ca2+ transient expressed by fs-α1SM701I + α1SΔ1–700 was indistinguishable from that of wt-α1S (t-test significance p = 0.671, see figure legend). To confirm expression of the C-terminus half of α1S in cells transfected with fs-α1S, we used the II-III loop polyclonal antibody SKI [34] directed against epitope Ala739-Ile752 which is downstream from Met701. Fig. 6C shows that the II-III loop antibody recognized the C-terminus half when cells were transfected with fs-α1S but not when myotubes were transfected with fs-α1SM701I. The C-terminal protein migrated with a molecular weight of approximately 126 KDa which is consistent with the theoretical molecular weight of 132 KDa. Finally, Fig. 6D shows that fs-α1SM701I was abundantly expressed in myotubes in the absence or presence of the C-terminal fragment. This indicated that the absence of EC coupling observed in myotubes expressing fs-α1SM701I was not due the production of a labile protein. In summary, the recovery of EC coupling by coexpression of two functionally inactive proteins (Fig. 5) taken together with the immunoblots (Fig. 6C) favor the explanation that 1) fs-α1S recovers DHPR function by virtue of expressing two complementary fragments of α1S and 2) the expression of the C-terminal half of α1S by fs-α1S is likely to occur by leaky ribosomal scanning.


Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (alpha1S) expressing two complementary protein fragments.

Ahern CA, Vallejo P, Mortenson L, Coronado R - BMC Physiol. (2001)

EC coupling generated by two complementary fragments of α1S. Line scans (horizontal dimension is 2.05 seconds) of fluo-4 fluorescence show Ca2+ transients in response to the indicated 50-ms depolarization from a holding potential of -40 mV. Trace of integrated fluorescence in ΔF/Fo units is shown for each line scan. Each set of depolarizations is from a separate dysgenic myotube expressing the α1S construct(s) indicated at the top of each column. A 16-color calibration bar in ΔF/Fo units is included in Fig. 3 for visual reference.
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Related In: Results  -  Collection

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Figure 5: EC coupling generated by two complementary fragments of α1S. Line scans (horizontal dimension is 2.05 seconds) of fluo-4 fluorescence show Ca2+ transients in response to the indicated 50-ms depolarization from a holding potential of -40 mV. Trace of integrated fluorescence in ΔF/Fo units is shown for each line scan. Each set of depolarizations is from a separate dysgenic myotube expressing the α1S construct(s) indicated at the top of each column. A 16-color calibration bar in ΔF/Fo units is included in Fig. 3 for visual reference.
Mentions: The EC coupling recovered by fs-α1S could be due either to the activity of the N-terminal half of α1S alone or to protein-protein complementation between the N-terminal half and a fragment expressing the C-terminal half of α1S. The C-terminal half of α1S could have been translated by the fs-α1S expression vector if the ATG codon (Met701), which is downstream from the TGA termination codon and is in-frame with the wild-type message (Fig 1B) served as open reading frame for translation of the second half of the wt message. Although this would be unusual, the fact that the codon for Met701 is only 25 bases downstream from the termination codon could have substantially increased the possibility of a re-start of the translation of the second half of the message at Met701. This phenomenon has been described in eukaryotic cells and in viral-infected mammalian cells and is known as translation by leaky ribosomal scanning [32,33]. To test this explanation, the presumptive restart condon, Met701, was mutated to Ile701 in the fs-α1S template. If fs-α1S recovered EC coupling by virtue of expressing a single protein fragment, then fs-α1SM701I should also recover EC coupling since the mutation was introduced downstream from the stop codon. Fig. 5 shows that this was not the case. Fs-α1SM701I did not recover Ca2+ transients in 9 of 9 tested cells, consistent with leaky ribosomal scanning. As a positive control, we coexpressed fs-α1SM701I and the C-terminus half of α1S, namely α1SΔ1–700, cloned into a separate pSG5 vector. The results in Fig. 5 indicated that α1SΔ1–700 alone was inactive. However, when myotubes were cotransfected with fs-α1SM701I and α1SΔ1–700, each in a separate pSG5 vector, there was a robust recovery of Ca2+ transients in 5 of 5 cells. Fig. 6A shows fluorescence vs. voltage relationships for the fs-α1SM701I mutant and for this mutant coexpressed with α1SΔ1–700. The combined expression of the two complementary fragments of α1S resulted in a robust recovery of EC coupling with sigmoidal Ca2+ release vs. voltage characteristics. A summary of the maximum fluorescence during the Ca2+ transient in response to a depolarization to +90 mV is shown in Fig. 6B. The magnitude of the Ca2+ transient expressed by fs-α1SM701I + α1SΔ1–700 was indistinguishable from that of wt-α1S (t-test significance p = 0.671, see figure legend). To confirm expression of the C-terminus half of α1S in cells transfected with fs-α1S, we used the II-III loop polyclonal antibody SKI [34] directed against epitope Ala739-Ile752 which is downstream from Met701. Fig. 6C shows that the II-III loop antibody recognized the C-terminus half when cells were transfected with fs-α1S but not when myotubes were transfected with fs-α1SM701I. The C-terminal protein migrated with a molecular weight of approximately 126 KDa which is consistent with the theoretical molecular weight of 132 KDa. Finally, Fig. 6D shows that fs-α1SM701I was abundantly expressed in myotubes in the absence or presence of the C-terminal fragment. This indicated that the absence of EC coupling observed in myotubes expressing fs-α1SM701I was not due the production of a labile protein. In summary, the recovery of EC coupling by coexpression of two functionally inactive proteins (Fig. 5) taken together with the immunoblots (Fig. 6C) favor the explanation that 1) fs-α1S recovers DHPR function by virtue of expressing two complementary fragments of α1S and 2) the expression of the C-terminal half of α1S by fs-α1S is likely to occur by leaky ribosomal scanning.

Bottom Line: Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca2+ current.In these cases, function is recovered by expression of complementary protein fragments from the same cDNA.DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca2+ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706, USA. cahern@physiology.wisc.edu

ABSTRACT

Background: The L-type Ca2+ channel formed by the dihydropyridine receptor (DHPR) of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1). This channel-to-channel coupling is essential for Ca2+ signaling but poorly understood. We characterized a single-base frame-shift mutant of alpha1S, the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC) coupling by virtue of expressing two complementary hemi-Ca2+ channel fragments.

Results: Functional analysis of cDNA transfected dysgenic myotubes lacking alpha1S were carried out using voltage-clamp, confocal Ca2+ indicator fluoresence, epitope immunofluorescence and immunoblots of expressed proteins. The frame-shift mutant (fs-alpha1S) expressed the N-terminal half of alpha1S (M1 to L670) and the C-terminal half starting at M701 separately. The C-terminal fragment was generated by an unexpected restart of translation of the fs-alpha1S message at M701 and was eliminated by a M701I mutation. Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca2+ current.

Discussion: A premature stop codon in the II-III loop may not necessarily cause a loss of DHPR function due to a restart of translation within the II-III loop, presumably by a mechanism involving leaky ribosomal scanning. In these cases, function is recovered by expression of complementary protein fragments from the same cDNA. DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca2+ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel.

Show MeSH
Related in: MedlinePlus