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Cooperative regulation of Ca(v)1.2 channels by intracellular Mg(2+), the proximal C-terminal EF-hand, and the distal C-terminal domain.

Brunet S, Scheuer T, Catterall WA - J. Gen. Physiol. (2009)

Bottom Line: We confirmed that increased Mg(i) reduces peak ionic currents and increases VDI of Ca(v)1.2 channels in ventricular myocytes and in transfected cells when measured with Na(+) as permeant ion.The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg(i) binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue.Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca(v)1.2 channels by Mg(i), the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195, USA. sbrunet@u.washington.edu

ABSTRACT
L-type Ca(2+) currents conducted by Ca(v)1.2 channels initiate excitation-contraction coupling in cardiac myocytes. Intracellular Mg(2+) (Mg(i)) inhibits the ionic current of Ca(v)1.2 channels. Because Mg(i) is altered in ischemia and heart failure, its regulation of Ca(v)1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg(i) on voltage-dependent inactivation (VDI) of Ca(v)1.2 channels using Na(+) as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca(2+)-dependent inactivation process. We confirmed that increased Mg(i) reduces peak ionic currents and increases VDI of Ca(v)1.2 channels in ventricular myocytes and in transfected cells when measured with Na(+) as permeant ion. The increased rate and extent of VDI caused by increased Mg(i) were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg(i) to the EF-hand (K(D) approximately 0.9 mM) near the resting level of Mg(i) in ventricular myocytes. VDI was more rapid for L-type Ca(2+) currents in ventricular myocytes than for Ca(v)1.2 channels in transfected cells. Coexpression of Ca(v)beta(2b) subunits and formation of an autoinhibitory complex of truncated Ca(v)1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca(v)1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg(i) binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca(v)1.2 channels by Mg(i), the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

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Effects of the distal C terminus on modulation of VDI by Mgi. Effect of Mgi on VDI in mean normalized current traces recorded from expression of (A) full-length CaV1.2, (B) truncated CaV1.2Δ1821, and (C) CaV1.2Δ1821 coexpressed with distal1822–2171. In A, n = 10 and 8 for 0.26 and 7.2 mM Mgi, respectively. In B, n = 10 and 7. In C, n = 5, 16, and 11 for 0.1, 0.26, and 7.2 mM Mgi, respectively.
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fig8: Effects of the distal C terminus on modulation of VDI by Mgi. Effect of Mgi on VDI in mean normalized current traces recorded from expression of (A) full-length CaV1.2, (B) truncated CaV1.2Δ1821, and (C) CaV1.2Δ1821 coexpressed with distal1822–2171. In A, n = 10 and 8 for 0.26 and 7.2 mM Mgi, respectively. In B, n = 10 and 7. In C, n = 5, 16, and 11 for 0.1, 0.26, and 7.2 mM Mgi, respectively.

Mentions: Because both the distal C terminus and Mgi enhance inactivation of CaV1.2 channels, we examined the impact of the distal C terminus on the Mgi modulation of VDI of truncated CaV1.2 channels. As in previous work, Mgi enhanced the inactivation of the full-length CaV1.2 channel (Fig. 8 A) (Brunet et al., 2005a), with r1000 = 0.54 ± 0.03 for 0.26 mM versus 0.15 ± 0.03 for 7.2 mM Mgi (P < 0.001). Similar to the Mgi effect on full-length channels, Mgi also enhanced VDI of CaV1.2Δ1821 (Fig. 8 B). The r1000 value was 0.54 ± 0.03 at 0.26 mM Mgi versus 0.10 ± 0.05 (P < 0.001) for 7.2 mM Mgi. In contrast, when distal1822–2171 was coexpressed with CaV1.2Δ1821 channels, 7.2 mM Mgi did not significantly enhance VDI of CaV1.2 (Fig. 8 C; 0.26 mM, r1000 = 0.28 ± 0.03; 7.2 mM, r1000 = 0.25 ± 0.03; P = 0.52). To determine whether reduced Mgi regulates this autoinhibitory complex, a lower Mgi concentration (0.1 mM) was tested. The dialysis of 0.1 mM Mgi reduced inactivation (r1000 = 0.60 ± 0.05; P < 0.01) compared with 0.26 and 7.2 mM Mgi (Fig. 8 C). This result suggests that the noncovalent interaction of the distal C terminus with the CaV1.2 channel enhances regulation of VDI by Mgi, possibly by increasing the affinity for binding of Mgi to the proximal C-terminal EF-hand.


Cooperative regulation of Ca(v)1.2 channels by intracellular Mg(2+), the proximal C-terminal EF-hand, and the distal C-terminal domain.

Brunet S, Scheuer T, Catterall WA - J. Gen. Physiol. (2009)

Effects of the distal C terminus on modulation of VDI by Mgi. Effect of Mgi on VDI in mean normalized current traces recorded from expression of (A) full-length CaV1.2, (B) truncated CaV1.2Δ1821, and (C) CaV1.2Δ1821 coexpressed with distal1822–2171. In A, n = 10 and 8 for 0.26 and 7.2 mM Mgi, respectively. In B, n = 10 and 7. In C, n = 5, 16, and 11 for 0.1, 0.26, and 7.2 mM Mgi, respectively.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2717695&req=5

fig8: Effects of the distal C terminus on modulation of VDI by Mgi. Effect of Mgi on VDI in mean normalized current traces recorded from expression of (A) full-length CaV1.2, (B) truncated CaV1.2Δ1821, and (C) CaV1.2Δ1821 coexpressed with distal1822–2171. In A, n = 10 and 8 for 0.26 and 7.2 mM Mgi, respectively. In B, n = 10 and 7. In C, n = 5, 16, and 11 for 0.1, 0.26, and 7.2 mM Mgi, respectively.
Mentions: Because both the distal C terminus and Mgi enhance inactivation of CaV1.2 channels, we examined the impact of the distal C terminus on the Mgi modulation of VDI of truncated CaV1.2 channels. As in previous work, Mgi enhanced the inactivation of the full-length CaV1.2 channel (Fig. 8 A) (Brunet et al., 2005a), with r1000 = 0.54 ± 0.03 for 0.26 mM versus 0.15 ± 0.03 for 7.2 mM Mgi (P < 0.001). Similar to the Mgi effect on full-length channels, Mgi also enhanced VDI of CaV1.2Δ1821 (Fig. 8 B). The r1000 value was 0.54 ± 0.03 at 0.26 mM Mgi versus 0.10 ± 0.05 (P < 0.001) for 7.2 mM Mgi. In contrast, when distal1822–2171 was coexpressed with CaV1.2Δ1821 channels, 7.2 mM Mgi did not significantly enhance VDI of CaV1.2 (Fig. 8 C; 0.26 mM, r1000 = 0.28 ± 0.03; 7.2 mM, r1000 = 0.25 ± 0.03; P = 0.52). To determine whether reduced Mgi regulates this autoinhibitory complex, a lower Mgi concentration (0.1 mM) was tested. The dialysis of 0.1 mM Mgi reduced inactivation (r1000 = 0.60 ± 0.05; P < 0.01) compared with 0.26 and 7.2 mM Mgi (Fig. 8 C). This result suggests that the noncovalent interaction of the distal C terminus with the CaV1.2 channel enhances regulation of VDI by Mgi, possibly by increasing the affinity for binding of Mgi to the proximal C-terminal EF-hand.

Bottom Line: We confirmed that increased Mg(i) reduces peak ionic currents and increases VDI of Ca(v)1.2 channels in ventricular myocytes and in transfected cells when measured with Na(+) as permeant ion.The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg(i) binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue.Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca(v)1.2 channels by Mg(i), the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195, USA. sbrunet@u.washington.edu

ABSTRACT
L-type Ca(2+) currents conducted by Ca(v)1.2 channels initiate excitation-contraction coupling in cardiac myocytes. Intracellular Mg(2+) (Mg(i)) inhibits the ionic current of Ca(v)1.2 channels. Because Mg(i) is altered in ischemia and heart failure, its regulation of Ca(v)1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg(i) on voltage-dependent inactivation (VDI) of Ca(v)1.2 channels using Na(+) as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca(2+)-dependent inactivation process. We confirmed that increased Mg(i) reduces peak ionic currents and increases VDI of Ca(v)1.2 channels in ventricular myocytes and in transfected cells when measured with Na(+) as permeant ion. The increased rate and extent of VDI caused by increased Mg(i) were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg(i) to the EF-hand (K(D) approximately 0.9 mM) near the resting level of Mg(i) in ventricular myocytes. VDI was more rapid for L-type Ca(2+) currents in ventricular myocytes than for Ca(v)1.2 channels in transfected cells. Coexpression of Ca(v)beta(2b) subunits and formation of an autoinhibitory complex of truncated Ca(v)1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca(v)1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg(i) binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca(v)1.2 channels by Mg(i), the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

Show MeSH
Related in: MedlinePlus