Succinate

Mice were killed, the eyes enucleated, and whole retinas removed from eye cups under infra-red illumination. Small pieces of retina were dissected in a drop of chilled Locke's solution (112.5 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl₂, 1.2 mM CaCl₂, 10 mM HEPES, 0.02 mM EDTA, 20 mM NaHCO₃, 3 mM Na₂-succinate, 0.5 mM Na-glutamate, 10 mM glucose), and placed into a recording chamber. The chamber was continuously refreshed with Locke's solution, pH 7.4, equilibrated with 95% O₂/5% CO₂, and maintained at 35–37°C with a heating system designed for microscopy (ALA Scientific). Using silanized suction pipettes, we recorded from photoreceptors embedded in 50–100-μm diameter slices of retina exclusively in the “OS out” configuration (Nikonov et al., 2005 (link)); in this effort several nuclei and conjoined “inner segment” tissue were intentionally drawn into the pipette. Once the tissue was drawn into the pipette, responses were evoked with calibrated flashes of light delivered under control of a customized LabView (National Instruments) interface. The optical system in the configuration used for these experiments has two stimulation channels: the light source in one channel is a tungsten-halogen lamp, and in the second a xenon flash lamp that delivers ∼20-μs pulses. Experiments with WT mouse retinal slices required the use of steady illumination to suppress rod activity, and the tungsten-halogen channel was employed for this purpose.
The “inner segment” limb of the rod and cone circulating current is an outward membrane current, carried primarily by K⁺ channels; light responses recorded from inner segment membranes are thus recorded by the amplifier as negative-going, resulting from the suppression of the outward membrane current as the cell hyperpolarizes toward the K⁺ reversal potential. Here we will present all photocurrent responses in the conventional manner as positive-going. However, the actual sign (and direction) of the recorded membrane currents will be referred to as needed.
As the expression of mouse M-cone opsin in mice varies in a dorso-ventral gradient (Applebury et al., 2000 (link)), we developed a method that allows the dorsal or ventral region of the retina to be dissected under infrared illumination and used for suction pipette recordings (Nikonov et al., 2005 (link)). This method has played a critical role in the complete characterization of cone function in the WT mouse.

The constructed stoichiometric model of E. coli contains all presently known reactions in central carbon metabolism with 98 reactions and 60 metabolites (Supplementary Table I). To apply FBA, the reaction network was automatically translated into a stoichiometric matrix (Schilling and Palsson, 1998 (link)) by means of a parser program implemented in Matlab (MATLAB^®, version 7.0.0.19920 (R14), The MathWorks Inc., Natick, MA). Assuming steady-state mass balances, the production and consumption of each of the m intracellular metabolites M_i is balanced to yield
with
S corresponds to the stoichiometric matrix (m × n) and ν (n × 1) to the array of n metabolic fluxes with ν_i^lb as lower and ν_i^ub as upper bounds, respectively. The above equations represent the conservation law of mass that is fundamental to constraint-based modeling. For all herein presented stoichiometric analyses, maximization of biomass yield is synonymous to the frequently used maximization of growth rate objective (Price et al, 2004 (link)). This is because stoichiometric models are sets of linear balance equations that are inherently dimensionless, hence maximization of the biomass reaction optimizes the amount of product (i.e., the yield) rather than a time-dependent rate of formation. The P-to-O ratio constraint was implemented by omitting the energy-coupling NADH dehydrogenase I (Nuo), cytochrome oxidase bo3 (Cyo) and/or cytochrome oxidase bd (Cyd) components of the respiratory chain. For a ratio of unity, Cyd and Nuo were set equal to zero. Under anaerobic conditions, electron flow is only possible via the NADH oxidases Nuo or NADH dehydrogenase II (Ndh) to fumarate reductase (Frd), hence coupled to succinate fermentation. For nitrate respiration, the terminal oxidase nitrate reductase (Nar) was used instead of Cyd or Cyo (Unden and Bongaerts, 1997 ).
For the genome-scale analysis we used two recently reconstructed models of E. coli metabolism (Edwards and Palsson, 2000b (link); Reed et al, 2003 (link)). In silico growth was simulated on glucose minimal medium for all six environmental conditions. ADP remained unbalanced, since otherwise formation of adenosine would be carbon-limited. For the proton-balanced model of Reed et al (2003) (link), severe alternate optima occurred in central carbon metabolism given an unlimited proton exchange flux between the cell and the medium and a P-to-O ratio of 2, that is the upper bound of the biologically feasible range of P-to-O ratios (Unden and Bongaerts, 1997 ). To prevent the unlimited production of ATP equivalents through the ATPS4r reaction under this condition, all external protons involved in the respiratory chain and the transhydrogenase reaction were balanced (specifically, we balanced the external protons around the reactions ATPS4r, TDH2, CYTBD, CYTBO3, NO3R1, NO3R2, NADH6, NADH7, NADH8). A P-to-O ratio of 2 was implemented by assuming both the transport of four protons through CYTBO3 and NADH6 across the membrane and the diffusion of four protons through ATPS4r for the formation of one ATP equivalent.