BASIC MECHANISMS OF MEMBRANE TRANSPORT

BASIC MECHANISMS OF MEMBRANE TRANSPORT Transporters versus Channels. Both channels and transporters facilitate the membrane permeation of inorganic ions and organic compounds (Reuss, 2000). In general, channels have two primary states, open and closed, that are totally stochastic phenomena. Only in the open state do channels appear to act as pores for the selected ions, allowing their permeation across the plasma membrane. After opening, channels return to the closed state as a function of time. In contrast, a transporter forms an intermediate complex with the substrate (solute), and subsequently a conformational change in the transporter induces translocation of the substrates to the other side of the membrane. Therefore, there is a marked difference in turnover rates between channels and transporters. The turnover rate constants of typical channels are 106 to 108 s-1, whereas those of transporters are, at most, 101 to 103 s-1. Because a particular transporter forms intermediate complexes with specific compounds (referred to as substrates), transporter-mediated membrane transport is characterized by saturability and inhibition by substrate analogs, as described below. The basic mechanisms involved in solute transport across biological membranes include passive diffusion, facilitated diffusion, and active transport. Active transport can be further subdivided into primary and secondary active transport. These mechanisms are depicted in Figure 2-4 and described below. Passive Diffusion. Simple diffusion of a solute across the plasma membrane consists of three processes: partition from the aqueous to the lipid phase, diffusion across the lipid bilayer, and repartition into the aqueous phase on the opposite side. Diffusion of any solute (including drugs) occurs down an electrochemical potential gradient Dm of the solute, given by the equation: (2-1) where z is the charge valence of the solute, Em is the membrane voltage, F is the Faraday constant, R is the gas constant, T is the absolute temperature, C is the concentration of the solute inside (i) and outside (o) of the plasma membrane. The first term on the right side in Eq. (2-1) represents the electrical potential, and the second represents the chemical potential. For nonionized compounds, the flux J owing to simple diffusion is given by Fick's first law (permeability multiplied by the concentration difference). For ionized compounds, the difference in electrical potential across the plasma membrane needs to be taken into consideration. Assuming that the electrical field is constant, the flux is given by the Goldman-Hodgkin-Katz equation: (2-2) where P represents the permeability. The lipid and water solubility and the molecular weight and shape of the solute are determinants of the flux in passive diffusion; they are incorporated in the permeability constant P. The permeability constant positively correlates with the lipophilicity, determined by the partition between water and organic solvents, such as octanol, and is also related to the inverse of the square root of the molecular weight of the solute. At steady state, the electrochemical potentials of all compounds become equal across the plasma membrane. In the case of nonionized compounds, the steady-state concentrations are equal across the plasma membrane. For ionized compounds, however, the steady-state concentration ratio across the plasma membrane is affected by the membrane voltage and given by the Nernst equation (Eq. 2-3). (2-3) The membrane voltage is maintained by the ion gradients across the membrane. Facilitated Diffusion. Diffusion of ions and organic compounds across the plasma membrane may be facilitated by a membrane transporter. Facilitated diffusion is a form of transporter-mediated membrane transport that does not require energy input. Just as in passive diffusion, the transport of ionized and un-ionized compounds across the plasma membrane occurs down their electrochemical potential gradient. Therefore, steady state will be achieved when the electrochemical potentials of the compound on both sides of the membrane become equal. Active Transport. Active transport is the form of membrane transport that requires the input of energy. It is the transport of solutes against their electrochemical gradients, leading to the concentration of solutes on one side of the plasma membrane and the creation of potential energy in the electrochemical gradient formed. Active transport plays an important role in the uptake and efflux of drugs and other solutes. Depending on the driving force, active transport can be subdivided into primary and secondary active transport (Figure 2-4). Primary Active Transport. Membrane transport that directly couples with ATP hydrolysis is called primary active transport. ABC transporters are examples of primary active transporters. They contain one or two ATP binding cassettes and a highly conserved domain in the intracellular loop region that exhibits ATPase activity. In mammalian cells, primary active transporters mediate the unidirectional efflux of solutes across biological membranes. The molecular mechanism by which ATP hydrolysis is coupled to the active transport of substrates by ABC transporters is a subject of current investigation. Secondary Active Transport. In secondary active transport, the transport across a biological membrane of one solute S1 against its concentration gradient is energetically driven by the transport of another solute S2 in accordance with its concentration gradient. The driving force for this type of transport therefore is stored in the electrochemical potential created by the concentration difference of S2 across the plasma membrane. For example, an inwardly directed Na+concentration gradient across the plasma membrane is created by Na+,K+-ATPase. Under these conditions, inward movement of Na+ produces the energy to drive the movement of a substrate S1 against its concentration gradient by a secondary active transporter as in Na+/Ca2+ exchange. Depending on the transport direction of the solute, secondary active transporters are classified as either symporters or antiporters. Symporters, also termed cotransporters, transport S2 and S1 in the same direction, whereas antiporters, also termed exchangers, move their substrates in opposite directions (Figure 2-4). The free energy produced by one extracellular sodium ion (Na+) is given by the difference in the electrochemical potential across the plasma membrane: (2-4) The electrochemical potential of a nonionized compound Dms acquired from one extracellular Na+ is less than this value: (2-5) Therefore, the concentration ratio of the compound is given by the following equation: (2-6) Assuming that the concentration ratio of Na+ is 10 and that Em is -60 mV, ideally, symport of one nonionized organic compound with one Na+ ion can achieve a one hundredfold difference in the intracellular substrate concentration compared with the extracellular concentration. When more than one Na+ ion is coupled to the movement of the solute, a synergistic driving force results. For the case in which two Na+ ions are involved, (2-7) In this case, the substrate ideally is concentrated intracellularly one thousandfold relative to the extracellular space under the same conditions. The Na+/Ca2+ antiporter shows the effect of this dependence in the square of the concentration ratio of Na+; Ca2+ is transported from the cytosol (0.1 mM < [Ca2+] < 1 mM) to the plasma [Ca2+]free ~ 1.25 mM. KINETICS OF TRANSPORT The flux of a substrate (rate of transport) across a biological membrane via transporter-mediated processes is characterized by saturability. The relationship between the flux v and substrate concentration C in a transporter-mediated process is given by the Michaelis-Menten equation: (2-8) where Vmax is the maximum transport rate and is proportional to the density of transporters on the plasma membrane, and Km is the Michaelis constant, which represents the substrate concentration at which the flux is half the Vmax value. Km is an approximation of the dissociation constant of the substrate from the intermediate complex. When C is small compared with the Km value, the flux is increased in proportion to the substrate concentration (roughly linear with substrate concentration). However, if C is large compared with the Km value, the flux approaches a constant value (Vmax). The Km and Vmax values can be determined by examining the flux at different substrate concentrations. The Eadie-Hofstee plot often is used for graphical interpretation of saturation kinetics. Plotting clearance v/C on the y axis and flux v on the x axis gives a straight line. The y intercept represents the ratio Vmax/Km, and the slope of the line is the inverse of the Km value: (2-9) Involvement of multiple transporters with different Km values gives an Eadie-Hofstee plot that is curved. In algebraic terms, the Eadie-Hofstee plot of kinetic data is equivalent to the Scatchard plot of equilibrium binding data. Transporter-mediated membrane transport of a substrate is also characterized by inhibition by other compounds. The manner of inhibition can be categorized as one of three types: competitive, noncompetitive, and uncompetitive. Competitive inhibition occurs when substrates and inhibitors share a common binding site on the transporter, resulting in an increase in the apparent Km value in the presence of inhibitor. The flux of a substrate in the presence of a competitive inhibitor is (2-10) where I is the concentration of inhibitor, and Ki is the inhibition constant. Noncompetitive inhibition assumes that the inhibitor has an allosteric effect on the transporter, does not inhibit the formation of an intermediate complex of substrate and transporter, but does inhibit the subsequent translocation process. (2-11) Uncompetitive inhibition assumes that inhibitors can form a complex only with an intermediate complex of the substrate and transporter and inhibit subsequent translocation. (2-12)