Permeation and block of rat glur6 glutamate receptor channels by internal and external polyamines

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Abstract
Polyamine block of rat GluR6(Q) glutamate receptor channels was studied in outside‐out patches from transiently transfected HEK 293 cells. With symmetrical 150 mm Na+ and 30 μm internal spermine there was biphasic voltage dependence with 95% block at +40 mV but only 20% block at +140mV. Dose–inhibition analysis for external spermine also revealed biphasic block; the Ka at +40 mV (54 μm) was lower than at +80 (167μm) and –80 mV (78 μm). For internal polyamines relief from block was most pronounced for spermine, weaker for N‐(4‐hydroxyphenylpropanoyl)‐spermine (PPS), and virtually absent for philanthotoxin 343 (PhTX 343), suggesting that permeation of polyamines varies with cross‐sectional width (spermine, 0.44 nm; PPS, 0.70 nm; PhTX 343, 0.75 nm). With putrescine, spermidine, or spermine as sole external cations, 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 to Na+, PPut/PNa= 0.42) and –32.7 mV for spermidine (PSpd/PNa= 0.07). Currents carried by spermine were too small to analyse accurately in the majority of patches. Increasing [Na+]i from 44 to 330 mm had no effect on the potential for 50% block (V½) by 30 μm internal spermine; however, relief from block at positive membrane potentials increased with [Na+]i. In contrast, raising [Na+]o from 44 to 330 mm resulted in a depolarizing shift in V½, indicating a strong interaction between internal polyamines and external per meant ions. The Woodhull infinite barrier model of ion channel block adequately described the action of spermine at membrane potentials insufficient to produce relief from block. For 30 μm internal spermine such analysis gave Kd(0)= 2.5 μm, z θ= 1.97; block by 30 μm external spermine was weaker and less voltage dependent (Kd(0)= 37.8 μm and zδ= 0.55); δ and θ are electrical distances measured from the outside and inside, respectively. Fits of the Woodhull equation for a permeable blocker adequately described both onset and relief from block by spermine over a wide range of membrane potentials. However, the rate constants and zδ values estimated for block by internal spermine predicted much stronger external block than was measured experimentally, and vice versa. An Eyring rate theory model with two energy wells and three barriers explained qualitatively many characteristic features of the action of polyamines on GluPvs, including biphasic I–V relationships, weaker block by external than internal spermine and low permeability.