Background—Impairment of intracellular Ca²⁺ homeostasis and mitochondrial function has been implicated in the development of cardiomyopathy. Mitochondrial Ca²⁺ uptake is thought to be mediated by the Ca²⁺ uniporter (MCU) and a thus far speculative non-MCU pathway. However, the identity and properties of these pathways are a matter of intense debate, and possible functional alterations in diseased states have remained elusive.

Methods and Results—By patch clamping the inner membrane of mitochondria from nonfailing and failing human hearts, we have identified 2 previously unknown Ca²⁺-selective channels, referred to as mCa1 and mCa2. Both channels are voltage dependent but differ significantly in gating parameters. Compared with mCa2 channels, mCa1 channels exhibit a higher single-channel amplitude, shorter openings, a lower open probability, and 3 to 5 subconductance states. Similar to the MCU, mCa1 is inhibited by 200 nmol/L ruthenium 360, whereas mCa2 is insensitive to 200 nmol/L ruthenium 360 and reduced only by very high concentrations (10 µmol/L). Both mitochondrial Ca²⁺ channels are unaffected by blockers of other possibly Ca²⁺-conducting mitochondrial pores but were activated by spermine (1 mmol/L). Notably, activity of mCa1 and mCa2 channels is decreased in failing compared with nonfailing heart conditions, making them less effective for Ca²⁺ uptake and likely Ca²⁺-induced metabolism.

Conclusions—Thus, we conclude that the human mitochondrial Ca²⁺ uptake is mediated by these 2 distinct Ca²⁺ channels, which are functionally impaired in heart failure. Current properties reveal that the mCa1 channel underlies the human MCU and that the mCa2 channel is responsible for the ruthenium red–insensitive/low-sensitivity non-MCU–type mitochondrial Ca²⁺ uptake.