PERMEATION AND BLOCK OF RAT GLUR6 GLUTAMATE-RECEPTOR CHANNELS BY INTERNAL AND EXTERNAL POLYAMINES

1. Polyamine block of rat GauR6(Q) glutamate receptor channels was stu died in outside-out patches from transiently transfected HEK 293 cells . With symmetrical 150 mM Na+ and 30 mu M internal spermine there was biphasic voltage dependence with 95% block at +40 mV but only 20% bloc k at +140 mV. Dose-inhibition analysis for external spermine also reve aled biphasic block; the K-d at +40 mV (54 mu M) was lower than at +80 (167 mu M) and -80 mV (78 mu M). 2. For internal polyamines relief fr om block was most pronounced for spermine, weaker for N-(4-hydroxyphen ylpropanoyl)-spermine (PPS), and virtually absent for philanthotoxin 3 43 (PhTX 343), suggesting that permeation of polyamines varies with cr oss-sectional width(spermine, 0.44 nm; PPS, 0.70 nm; PhTX 343, 0.75 nm ). 3. With putrescine, spermidine, or spermine as sole external cation s, inward currents at -120 mV confirmed permeation of polyamines. For bi-ionic conditions with 90 mM polyamine and 150 mM Na-i(+), reversal potentials were -12.4 mV for putrescine (permeability ratio relative t o Na+, P-Put/P-Na = 0.42) and -32.7 mV for spermidine (P-Spd/P-Na = 0. 07). Currents carried by spermine were too small to analyse accurately in the majority of patches. 4. Increasing [Na+](i) from 44 to 330 mM had no effect on the potential for 50% block (V-1/2) by 30 mu M intern al spermine; however, relief from block at positive membrane potential s increased with [Na+](i). In contrast, raising [Na+](o) from 44 to 33 0 mM resulted in a depolarizing shift in V-1/2, indicating a strong in teraction between internal polyamines and external permeant ions. 5. T he Woodhull infinite barrier model of ion channel block adequately des cribed the action of spermine at membrane potentials insufficient to p roduce relief from block. For 30 mu M internal spermine such analysis gave K-d(0) = 2.5 mu M, z theta = 1.97; block by 30 mu M external sper mine was weaker and less voltage dependent (K-d(0) = 37.8 mu M and z d elta = 0.55); delta and theta are electrical distances measured from t he outside and inside, respectively. 6. Fits of the Woodhull equation for a permeable blocker adequately described both onset and relief fro m block by spermine over a wide range of membrane potentials. However, the rate constants and z delta values estimated for block by internal spermine predicted much stronger external block than was measured exp erimentally, and vice versa. 7. An Eyring rate theory model with two e nergy wells and three barriers explained qualitatively many characteri stic features of the action of polyamines on GluRs, including biphasic I-V relationships, weaker block by external than internal spermine an d low permeability.