L-type calcium channel as a cardiac oxygen sensor

Abstract

Acute oxygen sensing in the heart is thought to occur through redox regulation and phosphorylation of membrane channels. Here we report a novel O2-sensing mechanism involving the C-terminus of the L-type Ca2+ channel and regulated by PKA phosphorylation. In patch-clamped myocytes, oxygen deprivation decreased ICa within 40 s. The suppressive effect of anoxia was relieved by PKA-mediated phosphorylation only when Ca2+ was the charge carrier, whereas phosphorylated IBa remained sensitive to O2 withdrawal. Suppression of Ca2+ release by thapsigargin did not alter the response of ICa to anoxia, suggesting a mandatory role for Ca2+ influx and not Ca2+-induced Ca2+ release (CICR) in O2 regulation of the channel. Consistent with this idea, mutation of 80 amino acids in the Ca2+/CaM-binding domain of the recombinant alpha1C subunit that removes Ca2+ dependent inactivation (CDI) abolished O2 sensitivity of the channel. Our findings suggest that the Ca2+/CaM binding domain of the L-type Ca2+ may represent a molecular site for O2 sensing of the heart.

Figure 1

Acute anoxia suppresses I_Ca in cardiomyocytes and HEK293 cells expressing recombinant L-type Ca²⁺ channel. (A) Time course of I_ca suppression in response to extracellular O₂ deprivation in cardiac and HEK cells. (B) Quantitative representation of I_ca suppression in response to acute extracellular anoxia. (C–D) Representative traces of I_Ca in cardiac (C) and HEK cells (D) before and within 40 s of extracellular O₂ deprivation.

Figure 2

PKA phosphorylation protects against anoxic suppression of I_Ca. (A–C) Representative I–V traces (−60 to 80 mV) of cardiac Ca²⁺ current before and after removal of extracellular O₂ in myocytes dialyzed with 0 cAMP (A), 10 μM H-89 + 200 μM cAMP (B) and 200 μM cAMP internal solutions (C). (D–F) Cumulative representation of anoxic I_ca suppression under conditions shown in A–C.

Figure 3

Channel phosphorylation does not protect against the effects of O₂ deprivation when Ba²⁺ is the charge carrier through the channel. (A–C) Representative traces of phosphorylated I_Ca (A), phosphorylated I_Ca in presence of 1 uM thapsigargin, and phosphorylated I_Ba before and after oxygen deprivation in left ventricular myocytes. (D) Time course of current suppression in response to anoxia with Ca²⁺ and Ba²⁺ as charge carriers. (E) Quantitative representation of A–C (N = 7–9).

Figure 4

Mutation of 80 amino acids in the Ca²⁺/CaM binding domain of the C-terminus inhibits the effect of anoxia on Ba²⁺-carried currents through the channel. (A) Representative I_Ba trace in a HEK cell expressing the alpha_1C77 subunit of the L-type channel before and after removal of extracellular oxygen. (B) Amino acid sequence 1572–1651 of native L-type channel C-terminus. (C) Representative trace showing I_Ba response to anoxia in a HEK cell expressing the mutant alpha_1c86 subunit. (D) Amino acid sequence 1572–1651 in the mutant 86 channel C-tail.