Preferential regulation of rabbit cardiac L-type Ca2+ current by glycolytic derived ATP via a direct allosteric pathway

Abstract

1. The activity of Ca2+ channels is regulated by a number of mechanisms including direct allosteric modulation by intracellular ATP. Since ATP derived from glycolysis is preferentially used for membrane function, we hypothesized that glycolytic ATP also preferentially regulates cardiac L-type Ca2+ channels. 2. To test this hypothesis, peak L-type Ca2+ currents (ICa) were measured in voltage-clamped rabbit cardiomyocytes during glycolytic inhibition (2-deoxyglucose + pyruvate), oxidative inhibition (cyanide + glucose) or both (full metabolic inhibition; FMI). 3. A 10 min period of FMI resulted in a 40.0 % decrease in peak ICa at +10 mV (-5.1 +/- 0.6 versus -3.1 +/- 0.4 pA pF-1; n = 5, P < 0.01). Similar decreases in peak ICa were observed during glycolytic inhibition using 2-deoxyglucose (-6.2 +/- 0.2 versus -3.7 +/- 0.2 pA pF-1; n = 5, P < 0.01) or iodoacetamide (-6.7 +/- 0.3 versus -3.7 +/- 0.2 pA pF-1; n = 7, P < 0.01), but not following oxidative inhibition (-6.2 +/- 0.4 versus -6.4 +/- 0.3 pA pF-1; n = 5, n.s.). The reduction in ICa following glycolytic inhibition was not mediated by phosphate sequestration by 2-deoxyglucose or changes in intracellular pH. 4. Reductions in ICa were still observed when inorganic phosphate and creatine were included in the pipette, confirming a critical role for glycolysis in ICa regulation. 5. With 5 mM MgATP in the pipette during FMI, peak ICa decreased by only 18.4 % (-6.8 +/- 0.6 versus -5.5 +/- 0.3 pA pF-1; n = 4, P < 0.05), while inclusion of 5 mM MgAMP-PCP (beta,gamma-methyleneadenosine 5'-triphosphate, Mg2+ salt) completely prevented the decrease in peak ICa (-6.9 +/- 0.3 versus -6.5 +/- 0.3 pA pF-1; n = 5, n.s.). 6. Together, these results suggest that ICa is regulated by intracellular ATP derived from glycolysis and does not require hydrolysis of ATP. This regulation is expected to be energy conserving during periods of metabolic stress and myocardial ischaemia.

Figure 1. Effect of full metabolic inhibition on L-type Ca²⁺ current

A, once the whole-cell configuration was achieved, I_Ca was sampled at 0.2 Hz and I-V relationships were recorded at the times indicated by the open and filled circles. The typical raw traces (inset) indicate that I_Ca run-down was minor over the 10 min sampling period. B, mean I-V relationships showing that sampling I_Ca at 0.2 Hz for 10 min did not significantly reduce I_Ca under our conditions (n= 4). C, I_Ca was sampled for 5 min before the indicated interruption in order to record a control I-V relationship. This was followed by external wash-in (arrow) of 2-deoxyglucose (10 mM) and sodium cyanide (2 mM) (full metabolic inhibition, FMI). The raw traces (inset) demonstrate that I_Ca prior to FMI (open circle) was larger than I_Ca after FMI (filled circle). D, mean I-V relationships which show that, on average, 10 min of FMI resulted in a 40 % reduction of I_Ca at +10 mV (n= 5, P < 0.01).

Figure 2. Holding current in control and after various metabolic interventions

A, summary of holding current at -80 mV (▪) and -40 mV () for control, full metabolic inhibition (FMI), glycolytic inhibition (GI) and oxidative inhibition (OI). It is clearly evident that the holding current was similar at both -80 and -40 mV regardless of metabolic intervention (n= 5 for all groups). B, mean I-V relationships recorded 10 min after control conditions, FMI, GI and OI in the presence of 10 μM nifedipine were not significantly different (n= 5 for all groups).

Figure 3. Effect of glycolytic and oxidative inhibition on L-type Ca²⁺ current

A and C, in the whole-cell configuration, I_Ca was initially sampled for 5 min prior to the indicated interruption, in order to record a control I-V relationship. This was immediately followed by external wash-in (arrow) of 2-deoxyglucose (2-DG; 10 mM; A) or sodium cyanide (CN; 2 mM; C). The raw traces in A and C (insets) indicate that I_Ca was larger prior to (open circle) than after (filled circle) glycolytic inhibition and was not altered by oxidative phosphorylation, respectively. B and D, mean I-V relationships indicating that, on average, 10 min of glycolytic inhibition resulted in a 43 % reduction of I_Ca at +10 mV (n= 11, P < 0.01) and was completely reversed by 30 min wash-out of 2-deoxyglucose (n= 6) (B). On the other hand, 10 min of oxidative inhibition (n= 5) increased I_Ca by 16 % at 0 mV (P < 0.05) and did not alter I_Ca at +10 mV and more positive membrane potentials (D).

Figure 4. Effect of glycolytic inhibition on Ca²⁺ current with 2-deoxyglucose or iodoacetamide

A, a 10 min episode of glycolytic inhibition with 2-deoxyglucose (2-DG; 10 mM) in the presence of NaH₂PO₄ (P_i; 1.2 mM in bath and 3.0 mM in pipette) reduced I_Ca by 25.0 % (n= 4, P < 0.05). B, however, a 15 min exposure to 2-deoxyglucose in the presence of NaH₂PO₄ (1.2 mM in bath and 3.0 mM in pipette) resulted in a greater reduction of I_Ca (38.4 %, n= 4, P < 0.01). C, a functional creatine kinase shuttle (20 mM creatine (CR) in pipette) did not prevent reduction of I_Ca. D, when myocytes were exposed for 10 min to iodoacetamide (IAA; 100 μM) a 44.8 % reduction of I_Ca was observed (n= 7, P < 0.01), which was similar to the reduction observed following glycolytic inhibition by 2-deoxyglucose (Fig. 3B). E, reduction of I_Ca by IAA was greatly attenuated by the inclusion of 5 mM MgATP in the pipette solution (IAA-MgATP), as was also observed when 2-deoxyglucose was used to inhibit glycolysis.

Figure 5. Effect of full metabolic inhibition on Ca²⁺ current in the presence of high Hepes and/or NaHCO₃

To determine whether the reduction of I_Ca after FMI is related to alterations in pH_i, proton buffering conditions were improved; 50 mM Hepes was present in the bath and pipette solutions in A-D, plus 25 mM NaHCO₃ in the bath solution in C and D. A and C, in the whole-cell configuration, I_Ca was initially sampled for 5 min before the indicated interruption to record a control I-V relationship. This was followed with external wash-in (arrow) of 2-deoxyglucose (10 mM) and sodium cyanide (2 mM) (FMI). In A, it is evident that 50 mM Hepes in the bath and pipette solution did not prevent the reduction of I_Ca upon wash-in of metabolic inhibitors. C shows that 50 mM Hepes in the bath and pipette and 25 mM NaHCO₃ in the bath did not prevent the reduction of I_Ca upon wash-in of metabolic inhibitors. B and D, mean I-V relationships showing that, on average, metabolic inhibition with 50 mM Hepes only resulted in a 47.6 % reduction of I_Ca (B; n= 6, P < 0.01) compared with a 38.6 % reduction with 50 mM Hepes and 25 mM NaHCO₃ (D; n= 4, P < 0.01).

Figure 6. Effect of full metabolic inhibition on L-type calcium current in the presence of MgATP or MgAMP-PCP

MgATP (5 mM) or MgAMP-PCP (5 mM) was included in the pipette solution in A and B, and C and D, respectively. A and C, in the whole-cell configuration, I_Ca was initially sampled for 5 min before the indicated interruption in order to record a control I-V relationship. This was immediately followed by external wash-in (arrow) of 2-deoxyglucose (10 mM) and sodium cyanide (2 mM). The raw traces (insets) demonstrate that in the presence of MgATP (A) I_Ca prior to (open circle) was larger than I_Ca after (filled circle) FMI. However, when MgAMP-PCP was included in the pipette (C) FMI did not alter I_Ca. B and D, mean I-V relationships which show that, on average, 10 min of FMI resulted in a moderate but significant reduction of I_Ca at +10 mV with MgATP (B; n= 5, P < 0.05) and was unaltered with MgAMP-PCP (D; n= 5, n.s.).