UCP1: A transporter for H+ and fatty acid anions

Abstract

Adaptive thermogenesis regulates core body temperature, controls fat deposition, and contributes strongly to the overall energy balance. This process occurs in brown fat and requires uncoupling protein 1 (UCP1), an integral protein of the inner mitochondrial membrane. Classic biochemical studies revealed the general principle of adaptive thermogenesis: in the presence of long-chain fatty acids (FA), UCP1 increases the permeability of the inner mitochondrial membrane for H⁺, which makes brown fat mitochondria produce heat rather than ATP. However, the exact mechanism by which UCP1 increases the membrane H⁺ conductance in a FA-dependent manner has remained a fundamental unresolved question. Recently, the patch-clamp technique was successfully applied to the inner mitochondrial membrane of brown fat to directly characterize the H⁺ currents carried by UCP1. Based on the patch-clamp data, a new model of UCP1 operation was proposed. In brief, FA anions are transport substrates of UCP1, and UCP1 operates as an unusual FA anion/H⁺ symporter. Interestingly, in contrast to short-chain FA anions, long-chain FA anions cannot easily dissociate from UCP1 due to strong hydrophobic interactions established by their carbon tails, and a single long-chain FA participates in many H⁺ transport cycles. Therefore, in the presence of long-chain FA, endogenous activators of brown fat thermogenesis, UCP1 effectively operates as an H⁺ uniport. In addition to their transport function, long-chain FA competitively remove tonic inhibition of UCP1 by cytosolic purine nucleotides, thus enabling activation of the thermogenic H⁺ leak through UCP1 under physiological conditions.

Keywords: Brown fat; Fatty acid; Mitochondria; Thermogenesis; UCP1; Uncoupling.

Figure 1. Proposed models of UCP1 operation

H⁺ channel activated by allosteric binding of FA, OH⁻ channel activated by allosteric binding of FA, “H⁺ buffering” model, and “FA cycling” model are shown.

Figure 2. Mitochondrial patch-clamp

(A) Preparation of mitoplasts (vesicles of the whole IMM): (1) Mitochondria isolated from tissue lysate are subjected to low-pressure French press to rupture the OMM and release the IMM (mitoplasts are formed); (2) when mitoplasts are incubated in a KCl solution, the IMM is further released from the OMM and mitoplasts assume an 8-shaped form. Remnants of the OMM are attached to the IMM. (B) Formation of the gigaom seal between the glass patch pipette and the IMM (mitoplast attached configuration) is followed by break-in into the mitoplast to achieve a whole-mitoplast configuration for recording currents across the whole IMM. The IMM patch under the pipette is destroyed by high-amplitude voltage pulses (200–500 mV). After break in, the IMM is completely released from the OMM, and mitoplast assumes a round shape. An isolated 8-shaped mitoplast and a round mitoplast attached to the pipette after break-in are shown in the lower panel. (C) Patch-clamp recording from the whole IMM. FA-dependent H⁺ current via UCP1 before (black) and after (red) application of UCP1 inhibitor GDP into the bath. The voltage protocol used to induce the current is shown above the current traces. All voltages indicated are within the mitochondrial matrix in respect to the bath (cytosol).

Figure 3. Model of UCP1 operation based on the electrophysiological data

(A) Left panel: the mechanism of steady UCP1 current induced by short-chain low-pKa FA analogs added on the cytosolic face of the IMM. Short-chain FA are simply transported by UCP1 across the IMM. UCP1 has two conformation states, with substrate binding site exposed either to the cytosolic (c-state) or matrix (m-state) side of the IMM. To reflect the fact that long-chain FA anions cannot bind to UCP1 on the matrix side [19], the access to the substrate binding site in the m-state is shown narrower as compared to c-state. Right panel: an original trace of steady UCP1 current induced by short-chain low-pKa FA analogs. Voltage protocol is shown above. (B) Left panel: the mechanism of transient UCP1 current induced by long-chain low-pKa FA analogs added on the cytosolic face of the IMM. A long-chain FA⁻ analog is translocated by UCP1 similar to short-chain FA⁻, however the long carbon tail of FA⁻ establishes strong hydrophobic interaction with UCP1 to prevent FA⁻ dissociation. Thus, the negatively charged FA⁻ shuttles within the UCP1 translocation pathway in response to the transmembrane voltage, producing transient currents. These currents suggest that the UCP1 substrate binding site changes its position within the membrane during c-m conformation change. Right panel: an original trace of transient UCP1 current induced by long-chain low-pKa FA analogs. (C) Left panel: the mechanism of H⁺ current via UCP1 induced by regular long-chain FA added on the cytosolic face of the IMM. UCP1 operates as a symporter that transports one FA⁻ and one H⁺ per the transport cycle. The H⁺ and the FA⁻ are translocated by UCP1 upon a conformational change, and H⁺ is released on the opposite side of the IMM, while the FA⁻ stays associated with UCP1 due to the hydrophobic interactions established by its carbon tail. The FA⁻ anion then returns to initiate another H⁺ translocation cycle. Charge is translocated only in step 3 when the long chain FA anion returns without the H⁺. Right panel: an original trace of H⁺ current via UCP1 induced by regular long-chain FA.

Figure 4. Proposed substrate binding site of UCP1

Predicted SBS of UCP1 before (1) and after (2) binding of an FA anion and H⁺. Arginines R84, R183, and R277 are shown in blue, and D28 is shown as an open red circle in the water-filled cytosolic cavity. H⁺ is stabilized between the carboxylic headgroup of FA and D28 (2).

Figure 5. Proposed UCP1 conformational changes during the transport cycle

After initial binding of the anionic long-chain FA and an H⁺ to the SBS (1 and 2, for simplicity D28 and arginines are omitted), UCP1 changes its conformation and the FA penetrates into the area that separates the cytosolic cavity and the matrix, within the membrane electric field (occluded state, 3). The structure of the substrate-bound UCP1 (3) is flexible, and it spontaneously transitions between the cytosolic state (4) and the matrix state (5) to expose the FA headgroup to different sides of the IMM and accomplish H⁺ transport.