Modulation of recombinant human cardiac L‐type Ca2+ channel α1C subunits by redox agents and hypoxia

Abstract

1 Whole‐cell patch clamp recordings were used to investigate the modulation by reducing and oxidizing agents of recombinant human cardiac L‐type Ca²⁺ channel α_1C subunits stably expressed in human embryonic kidney (HEK 293) cells. 2 The oxidizing agents thimerosal (10 μM) and p‐chloromercuribenzene sulphonic acid (PCMBS; 2 μM to 2 mM) caused irreversible inhibition of Ca²⁺ channel currents. The reducing agent 1,4‐dithiothreitol (DTT; 2 mM) was without effect on Ca²⁺ channel currents, but reversed the inhibitory actions of thimerosal and PCMBS. 3 Ca²⁺ channel currents were also inhibited by pretreatment with the methanethiosulphonate compound (2‐aminoethyl)methanethiosulphonate (MTSEA, 2·5 mM), but were unaffected by identical pretreatment with (2‐sulphonatoethyl)methanethiosulphonate (MTSES, 10 mM). The effects of MTSEA could be fully reversed by DTT (2 mM). The degree of current inhibition caused by 200 μM PCMBS was not significantly affected by pretreatment with MTSEA, and following PCMBS treatment, MTSEA caused a similar degree of inhibition to that observed in cells that were not previously treated with PCMBS. These findings suggested that distinct thiol groups were modulated by these two agents. 4 Hypoxic inhibition of Ca²⁺ channel currents was unaffected by pretreatment of cells with MTSEA but was fully prevented by treatment with PCMBS. Our results indicate that distinct cysteine residues on the α_1C subunit can undergo redox modulation and in so doing alter channel function. Some, but not all, of these residues appear to be associated with the mechanism underlying inhibition of this channel by hypoxia.