Description

Pathway control states (synonymous with ETS substrate control states) are obtained in mitochondrial preparations (isolated mitochondria, permeabilized cells, permeabilized tissues, tissue homogenate) by depletion of endogenous substrates and addition of specific ETS substrate types to the mitochondrial respiration medium. Mitochondrial pathway control states have to be defined complementary to mitochondrial coupling control states. Coupling states (LEAK, OXPHOS, ETS) require electron transfer system competent substrate states, including oxygen supply. Categories of SUIT protocols are defined according to ETS substrate types. » MiPNet article

Reference: Gnaiger 2009 Int J Biochem Cell Biol, Gnaiger 2014 MitoPathways, Categories of SUIT protocols

MitoPedia concepts: MiP concept, Respiratory state, SUIT concept

MitoPedia methods: Respirometry

Pathway control states

Gnaiger E (2016) Pathway control states. Mitochondr Physiol Network 2016-08-12.

OROBOROS (2016) MiPNet

Abstract: Pathway control states are defined complementary to coupling control states in mitochondrial physiology.

• O2k-Network Lab: AT Innsbruck Gnaiger E

Labels:

Preparation: Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP

Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.

HRR: Theory

ETS competent pathway control states

Coupling states (LEAK, OXPHOS, ETS) require electron transfer system (ETS) competent pathway states based on external substrate supply, including sufficient oxygen supply. ETS competence of external substrates requires (i) transport of substrates across the inner mt-membrane or oxidation by dehydrogenases located on the outer face of the inner mt-membrane (e.g. glycerophosphate dehydrogenase complex, CGpDH), (ii) oxidation in the mt-matrix (TCA cycle dehydrogenases and other matrix dehydrogenases, e.g. mtGDH) or on the inner face of the inner mt-membrane (succinate dehydrogenase), (iii) oxidation of substrates without accumulation of inhibitory endproducts (e.g. oxaloacetate inhibiting succinate dehydrogenase; NADH and oxaloacetate inhibiting malate dehydrogenase), and (iv) electron transfer through the membrane-bound ETS (mETS). Endproducts must be either easily exported from the matrix across the inner mt-membrane (e.g. malate formed from succinate via fumarate), or metabolized in the TCA cycle (e.g. malate-derived oxaloacetate forming citrate in the presence of external pyruvate&malate).

Single pathway control states

Single pathway control states are pathway control states for selective entry of electron transfer into the Q-junction through one particular respiratory complex; for instance N-respiration through CI (PM; GM; PGM with or without malonic acid: Gnaiger 2014 MitoPathways Chapter 3), S-pathway control state with e-input into Q through CII, CIV (Tm: MiPNet06.06_Chemical O2 background).

Further details, see Categories of SUIT protocols.

Multiple pathway control states

Multiple pathway control states are pathway control states obtained in intact cells respiring on endogenous substrates or in media with physiological exogenous substrates, or designed for reconstitution of TCA cycle function in isolated mitochondria, permeabilized cells or permeabilized tissues. In all cases, electron flow converges at the Q-junction with multiple entry sites of NS-electron transfer through CI&II, FNS through FAO&CI&II, NSGp through CI&II&GpDH.

Further details » Categories of SUIT protocols

Pathway versus kinetic substrate control

Control by substrate type: pathway control states

A: Intact cells

Endogenous pathway control: In intact cells, ce, endogenous organic carbon substrates are mobilized in the cytosol as intermediary metabolites transported across the inner mitochondrial membrane and thus exerting control over mitochondrial respiration. If no organic carbon substrates are supplied in the incubation medium, then substrate control is entirely endogenous. Long-term incubation under such conditions leads to progressive depletion of endogenous substrates.
Exogenous pathway control: Cells are grown in complex culture media with a variety of organic carbon substrates, and different exogenous pathway control states are achieved by variation of these substrates. Long-term incubation in closed systems without exchange of culture medium leads to progressive depletion of exogenous substrates. Incubation of cells in simple media allows for sequential titration of specific carbon substrates (e.g. glucose or fructose; lactate or glutamate; fatty acids) for the study of exogenous pathway control of respiration.

B: Mitochondrial preparations

Specific substrate-inhibitor combinations are selected to establish pathway states for (i) stimulating defined segments of the electron transfer system, or (ii) reconstitution of TCA cycle function.

Pathway control states with electron gating: Specific substrate-inhibitor combinations are applied for selectively stimulating electron entry from N-junction substrates through CI, S-pathway control state through CII, or other substrates feeding additional branches converging at the Q-junction, particularly F-type (fatty acid oxidation and Gp (glycerophosphate). The most commonly applied pathway states select for N-electron input through Complex I (pyruvate&malate, PM; glutamate&malate, GM), S-pathway control state ([[Complex II]-pathway to Q]: succinate and rotenone, SRot), or Complex IV electron input (CIV: ascorbate&TMPD(Ama)).
Physiological pathway control states: Reconstitution of TCA cycle function requires NS-substrate combinations, such as PMS, GMS, GS, PGMS, or PGS, applied simultaneously without inhibitor of any respiratory complexes.

Control by substrate concentration: kinetic control states

Kinetic substrate or adenylate control: Kinetic studies with variation of a specific substrate (reduced substrate supplying electrons to the ETS; ADP, Pi; O₂; cytochrome c) are analyzed by kinetic functions (e.g. hyperbolic), yielding kinetic parameters, such as J_max, K_m', c₅₀ [µM], or p₅₀ [kPa].
Kinetic inhibitor control: Kinetic studies with variation of a specific inhibitor yield apparent kinetic constants, such as the K_I'.