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COOH-terminal truncated alpha(1S) subunits conduct current better than full-length dihydropyridine receptors.

Morrill JA, Cannon SC - J. Gen. Physiol. (2000)

Bottom Line: As in recordings from skeletal muscle, for heterologously expressed channels the peak inward Ba(2+) currents were small relative to Q(max).The truncated alpha(1SDeltaC) protein, however, supported much larger ionic currents than the full-length product.Our data also suggest that the carboxyl terminus of the alpha(1S) subunit modulates the coupling between charge movement and channel opening.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Skeletal muscle dihydropyridine (DHP) receptors function both as voltage-activated Ca(2+) channels and as voltage sensors for coupling membrane depolarization to release of Ca(2+) from the sarcoplasmic reticulum. In skeletal muscle, the principal or alpha(1S) subunit occurs in full-length ( approximately 10% of total) and post-transcriptionally truncated ( approximately 90%) forms, which has raised the possibility that the two functional roles are subserved by DHP receptors comprised of different sized alpha(1S) subunits. We tested the functional properties of each form by injecting oocytes with cRNAs coding for full-length (alpha(1S)) or truncated (alpha(1SDeltaC)) alpha subunits. Both translation products were expressed in the membrane, as evidenced by increases in the gating charge (Q(max) 80-150 pC). Thus, oocytes provide a robust expression system for the study of gating charge movement in alpha(1S), unencumbered by contributions from other voltage-gated channels or the complexities of the transverse tubules. As in recordings from skeletal muscle, for heterologously expressed channels the peak inward Ba(2+) currents were small relative to Q(max). The truncated alpha(1SDeltaC) protein, however, supported much larger ionic currents than the full-length product. These data raise the possibility that DHP receptors containing the more abundant, truncated form of the alpha(1S) subunit conduct the majority of the L-type Ca(2+) current in skeletal muscle. Our data also suggest that the carboxyl terminus of the alpha(1S) subunit modulates the coupling between charge movement and channel opening.

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Comparison of ionic current to gating charge movement in oocytes expressing α1S or α1SΔC plus the auxiliary subunits. (A–D) Qon versus voltage (left scale) and current versus voltage (right scale) measured in the same oocytes. (A) Data from six cells expressing α1S, β1a, α2δ, and γ. (B) Data from five cells expressing α1SΔC, β1a, α2δ, and γ. (C) Data from 10 cells expressing α1S, β1b, α2δ, and γ. (D) Data from nine cells expressing α1SΔC, β1b, α2δ, and γ. Experiments were performed as in Fig. 3. Qon, measured as described in Fig. 1, was used instead of Qoff in these experiments to avoid the possibility of contamination with residual Ba2+ tail current. The solid lines are the mean Boltzmann Q-V curves determined as in Fig. 2 for each set of data. In A, Qon, max = 140 ± 21.6 pC, V1/2Q = −12.3 ± 5.5 mV, kQ = 16.0 ± 1.7 mV, Imax = −8.5 ± 1.1 nA, and n = 6. In B, Qon, max = 165 ± 47.6 pC, V1/2Q = −9.2 ± 3.3 mV, kQ = 15.6 ± 2.3 mV, Imax = −53.0 ± 8.6 nA, and n = 5. The difference in Qon, max between the data in A and in B was not statistically significant (P = 0.63). In C, Qon, max = 80.1 ± 9.6 pC, V1/2Q = −24.6 ± 1.8 mV, kQ = 13.8 ± 1.4 mV, Imax = −7.3 ± 1.1 nA, and n = 10. In D, Qon, max = 125 ± 16.0 pC, V1/2Q = −27.4 ± 2.6 mV, kQ = 14.4 ± 1.8 mV, Imax = −68.8 ± 7.2 nA, and n = 6. The difference in Qon, max between C and D was statistically significant (P = 0.02). (E) Ratio of maximum Ba2+ current (Imax) to Qon, max in cells expressing α1SΔC or α1S plus auxiliary subunits. For each cell, the maximum inward current evoked by a 300-ms depolarization to +10 or +15 mV in 10-mM Ba2+ solution was divided by the value of Qon, max determined from the Boltzmann fit to the Qon versus voltage relationship. In cells expressing the β1a subunit, Imax/Qon, max was 0.06 ± 0.01 nA/pC when α1S was expressed and 0.37 ± 0.05 nA/pC when α1SΔC was expressed, a difference which was statistically significant (P = 4.8 × 10−6). In cells expressing the β1b subunit, Imax/Qon, max was 0.10 ± 0.01 nA/pC when α1S was expressed and 0.60 ± 0.09 nA/pC when α1SΔC was expressed, a difference which was also statistically significant (P = 6.2 × 10−5).
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Figure 4: Comparison of ionic current to gating charge movement in oocytes expressing α1S or α1SΔC plus the auxiliary subunits. (A–D) Qon versus voltage (left scale) and current versus voltage (right scale) measured in the same oocytes. (A) Data from six cells expressing α1S, β1a, α2δ, and γ. (B) Data from five cells expressing α1SΔC, β1a, α2δ, and γ. (C) Data from 10 cells expressing α1S, β1b, α2δ, and γ. (D) Data from nine cells expressing α1SΔC, β1b, α2δ, and γ. Experiments were performed as in Fig. 3. Qon, measured as described in Fig. 1, was used instead of Qoff in these experiments to avoid the possibility of contamination with residual Ba2+ tail current. The solid lines are the mean Boltzmann Q-V curves determined as in Fig. 2 for each set of data. In A, Qon, max = 140 ± 21.6 pC, V1/2Q = −12.3 ± 5.5 mV, kQ = 16.0 ± 1.7 mV, Imax = −8.5 ± 1.1 nA, and n = 6. In B, Qon, max = 165 ± 47.6 pC, V1/2Q = −9.2 ± 3.3 mV, kQ = 15.6 ± 2.3 mV, Imax = −53.0 ± 8.6 nA, and n = 5. The difference in Qon, max between the data in A and in B was not statistically significant (P = 0.63). In C, Qon, max = 80.1 ± 9.6 pC, V1/2Q = −24.6 ± 1.8 mV, kQ = 13.8 ± 1.4 mV, Imax = −7.3 ± 1.1 nA, and n = 10. In D, Qon, max = 125 ± 16.0 pC, V1/2Q = −27.4 ± 2.6 mV, kQ = 14.4 ± 1.8 mV, Imax = −68.8 ± 7.2 nA, and n = 6. The difference in Qon, max between C and D was statistically significant (P = 0.02). (E) Ratio of maximum Ba2+ current (Imax) to Qon, max in cells expressing α1SΔC or α1S plus auxiliary subunits. For each cell, the maximum inward current evoked by a 300-ms depolarization to +10 or +15 mV in 10-mM Ba2+ solution was divided by the value of Qon, max determined from the Boltzmann fit to the Qon versus voltage relationship. In cells expressing the β1a subunit, Imax/Qon, max was 0.06 ± 0.01 nA/pC when α1S was expressed and 0.37 ± 0.05 nA/pC when α1SΔC was expressed, a difference which was statistically significant (P = 4.8 × 10−6). In cells expressing the β1b subunit, Imax/Qon, max was 0.10 ± 0.01 nA/pC when α1S was expressed and 0.60 ± 0.09 nA/pC when α1SΔC was expressed, a difference which was also statistically significant (P = 6.2 × 10−5).

Mentions: Comparison of the mean Qon-V and I-V curves measured in oocytes expressing α1SΔC and α1S with either the β1a subunit (Fig. 4A and Fig. B) or the β1b subunit (C and D) emphasizes that while the full-length and truncated subunits are comparable voltage sensors, the truncated version is a much better conductor of ionic current. We quantified this functional distinction by calculating the ratio of maximum ionic current to maximum charge movement (Imax/Qon, max) for each oocyte (Fig. 4 E). Imax/Qon, max was sixfold greater for oocytes expressing α1SΔC than for oocytes expressing α1S when either the β1a or the β1b subunit was used; a difference that was highly statistically significant in each case (P = 4.8 × 10−6 and 6.2 × 10−5, respectively). This difference is a conservative estimate, since at least 90% of the current observed in α1S-injected oocytes was conducted by a DHP-insensitive α subunit, as mentioned above (data not shown). Imax/Qon, max was smaller for channels containing β1a than for channels containing β1b, reflecting both the somewhat smaller ionic currents and larger gating currents observed, on average, when β1a was injected. As was also seen in the separate experiments of Fig. 2, truncation of the α1S increased the size of gating currents significantly in the presence of the β1b subunit (P = 0.02), but not in the presence of the β1a subunit (P = 0.63).


COOH-terminal truncated alpha(1S) subunits conduct current better than full-length dihydropyridine receptors.

Morrill JA, Cannon SC - J. Gen. Physiol. (2000)

Comparison of ionic current to gating charge movement in oocytes expressing α1S or α1SΔC plus the auxiliary subunits. (A–D) Qon versus voltage (left scale) and current versus voltage (right scale) measured in the same oocytes. (A) Data from six cells expressing α1S, β1a, α2δ, and γ. (B) Data from five cells expressing α1SΔC, β1a, α2δ, and γ. (C) Data from 10 cells expressing α1S, β1b, α2δ, and γ. (D) Data from nine cells expressing α1SΔC, β1b, α2δ, and γ. Experiments were performed as in Fig. 3. Qon, measured as described in Fig. 1, was used instead of Qoff in these experiments to avoid the possibility of contamination with residual Ba2+ tail current. The solid lines are the mean Boltzmann Q-V curves determined as in Fig. 2 for each set of data. In A, Qon, max = 140 ± 21.6 pC, V1/2Q = −12.3 ± 5.5 mV, kQ = 16.0 ± 1.7 mV, Imax = −8.5 ± 1.1 nA, and n = 6. In B, Qon, max = 165 ± 47.6 pC, V1/2Q = −9.2 ± 3.3 mV, kQ = 15.6 ± 2.3 mV, Imax = −53.0 ± 8.6 nA, and n = 5. The difference in Qon, max between the data in A and in B was not statistically significant (P = 0.63). In C, Qon, max = 80.1 ± 9.6 pC, V1/2Q = −24.6 ± 1.8 mV, kQ = 13.8 ± 1.4 mV, Imax = −7.3 ± 1.1 nA, and n = 10. In D, Qon, max = 125 ± 16.0 pC, V1/2Q = −27.4 ± 2.6 mV, kQ = 14.4 ± 1.8 mV, Imax = −68.8 ± 7.2 nA, and n = 6. The difference in Qon, max between C and D was statistically significant (P = 0.02). (E) Ratio of maximum Ba2+ current (Imax) to Qon, max in cells expressing α1SΔC or α1S plus auxiliary subunits. For each cell, the maximum inward current evoked by a 300-ms depolarization to +10 or +15 mV in 10-mM Ba2+ solution was divided by the value of Qon, max determined from the Boltzmann fit to the Qon versus voltage relationship. In cells expressing the β1a subunit, Imax/Qon, max was 0.06 ± 0.01 nA/pC when α1S was expressed and 0.37 ± 0.05 nA/pC when α1SΔC was expressed, a difference which was statistically significant (P = 4.8 × 10−6). In cells expressing the β1b subunit, Imax/Qon, max was 0.10 ± 0.01 nA/pC when α1S was expressed and 0.60 ± 0.09 nA/pC when α1SΔC was expressed, a difference which was also statistically significant (P = 6.2 × 10−5).
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Figure 4: Comparison of ionic current to gating charge movement in oocytes expressing α1S or α1SΔC plus the auxiliary subunits. (A–D) Qon versus voltage (left scale) and current versus voltage (right scale) measured in the same oocytes. (A) Data from six cells expressing α1S, β1a, α2δ, and γ. (B) Data from five cells expressing α1SΔC, β1a, α2δ, and γ. (C) Data from 10 cells expressing α1S, β1b, α2δ, and γ. (D) Data from nine cells expressing α1SΔC, β1b, α2δ, and γ. Experiments were performed as in Fig. 3. Qon, measured as described in Fig. 1, was used instead of Qoff in these experiments to avoid the possibility of contamination with residual Ba2+ tail current. The solid lines are the mean Boltzmann Q-V curves determined as in Fig. 2 for each set of data. In A, Qon, max = 140 ± 21.6 pC, V1/2Q = −12.3 ± 5.5 mV, kQ = 16.0 ± 1.7 mV, Imax = −8.5 ± 1.1 nA, and n = 6. In B, Qon, max = 165 ± 47.6 pC, V1/2Q = −9.2 ± 3.3 mV, kQ = 15.6 ± 2.3 mV, Imax = −53.0 ± 8.6 nA, and n = 5. The difference in Qon, max between the data in A and in B was not statistically significant (P = 0.63). In C, Qon, max = 80.1 ± 9.6 pC, V1/2Q = −24.6 ± 1.8 mV, kQ = 13.8 ± 1.4 mV, Imax = −7.3 ± 1.1 nA, and n = 10. In D, Qon, max = 125 ± 16.0 pC, V1/2Q = −27.4 ± 2.6 mV, kQ = 14.4 ± 1.8 mV, Imax = −68.8 ± 7.2 nA, and n = 6. The difference in Qon, max between C and D was statistically significant (P = 0.02). (E) Ratio of maximum Ba2+ current (Imax) to Qon, max in cells expressing α1SΔC or α1S plus auxiliary subunits. For each cell, the maximum inward current evoked by a 300-ms depolarization to +10 or +15 mV in 10-mM Ba2+ solution was divided by the value of Qon, max determined from the Boltzmann fit to the Qon versus voltage relationship. In cells expressing the β1a subunit, Imax/Qon, max was 0.06 ± 0.01 nA/pC when α1S was expressed and 0.37 ± 0.05 nA/pC when α1SΔC was expressed, a difference which was statistically significant (P = 4.8 × 10−6). In cells expressing the β1b subunit, Imax/Qon, max was 0.10 ± 0.01 nA/pC when α1S was expressed and 0.60 ± 0.09 nA/pC when α1SΔC was expressed, a difference which was also statistically significant (P = 6.2 × 10−5).
Mentions: Comparison of the mean Qon-V and I-V curves measured in oocytes expressing α1SΔC and α1S with either the β1a subunit (Fig. 4A and Fig. B) or the β1b subunit (C and D) emphasizes that while the full-length and truncated subunits are comparable voltage sensors, the truncated version is a much better conductor of ionic current. We quantified this functional distinction by calculating the ratio of maximum ionic current to maximum charge movement (Imax/Qon, max) for each oocyte (Fig. 4 E). Imax/Qon, max was sixfold greater for oocytes expressing α1SΔC than for oocytes expressing α1S when either the β1a or the β1b subunit was used; a difference that was highly statistically significant in each case (P = 4.8 × 10−6 and 6.2 × 10−5, respectively). This difference is a conservative estimate, since at least 90% of the current observed in α1S-injected oocytes was conducted by a DHP-insensitive α subunit, as mentioned above (data not shown). Imax/Qon, max was smaller for channels containing β1a than for channels containing β1b, reflecting both the somewhat smaller ionic currents and larger gating currents observed, on average, when β1a was injected. As was also seen in the separate experiments of Fig. 2, truncation of the α1S increased the size of gating currents significantly in the presence of the β1b subunit (P = 0.02), but not in the presence of the β1a subunit (P = 0.63).

Bottom Line: As in recordings from skeletal muscle, for heterologously expressed channels the peak inward Ba(2+) currents were small relative to Q(max).The truncated alpha(1SDeltaC) protein, however, supported much larger ionic currents than the full-length product.Our data also suggest that the carboxyl terminus of the alpha(1S) subunit modulates the coupling between charge movement and channel opening.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Skeletal muscle dihydropyridine (DHP) receptors function both as voltage-activated Ca(2+) channels and as voltage sensors for coupling membrane depolarization to release of Ca(2+) from the sarcoplasmic reticulum. In skeletal muscle, the principal or alpha(1S) subunit occurs in full-length ( approximately 10% of total) and post-transcriptionally truncated ( approximately 90%) forms, which has raised the possibility that the two functional roles are subserved by DHP receptors comprised of different sized alpha(1S) subunits. We tested the functional properties of each form by injecting oocytes with cRNAs coding for full-length (alpha(1S)) or truncated (alpha(1SDeltaC)) alpha subunits. Both translation products were expressed in the membrane, as evidenced by increases in the gating charge (Q(max) 80-150 pC). Thus, oocytes provide a robust expression system for the study of gating charge movement in alpha(1S), unencumbered by contributions from other voltage-gated channels or the complexities of the transverse tubules. As in recordings from skeletal muscle, for heterologously expressed channels the peak inward Ba(2+) currents were small relative to Q(max). The truncated alpha(1SDeltaC) protein, however, supported much larger ionic currents than the full-length product. These data raise the possibility that DHP receptors containing the more abundant, truncated form of the alpha(1S) subunit conduct the majority of the L-type Ca(2+) current in skeletal muscle. Our data also suggest that the carboxyl terminus of the alpha(1S) subunit modulates the coupling between charge movement and channel opening.

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Related in: MedlinePlus