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

Abstract

1. Polyamine block of rat GluR6(Q) glutamate receptor channels was studied in outside-out patches from transiently transfected HEK 293 cells. With symmetrical 150 mÒ Na¤ and 30 ìÒ internal spermine there was biphasic voltage dependence with 95% block at +40 mV but only 20% block at +140 mV. Dose-inhibition analysis for external spermine also revealed biphasic block; the Kd at +40 mV (54 ìÒ) was lower than at +80 (167 ìÒ) and −80 mV (78 ìÒ). 2. 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). 3. With putrescine, spermidine, or spermine as sole external cations, inward currents at −120 mV confirmed permeation of polyamines. For bi-ionic conditions with 90 mÒ polyamine and 150 mÒ Na¸, reversal potentials were −12·4 mV for putrescine (permeability ratio relative to Na¤, PPutÏPNa = 0·42) and −32·7 mV for spermidine (PSpdÏPNa = 0·07). Currents carried by spermine were too small to analyse accurately in the majority of patches. 4. Increasing [Na¤]é from 44 to 330 mÒ had no effect on the potential for 50% block (V½) by 30 ìÒ internal spermine; however, relief from block at positive membrane potentials increased with [Na¤]é. In contrast, raising [Na¤]ï from 44 to 330 mÒ resulted in a depolarizing shift in V½, indicating a strong interaction between internal polyamines and external permeant ions. 5. 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 ìÒ internal spermine such analysis gave Kd(0) = 2·5 ìÒ, zè = 1·97; block by 30 ìÒ external spermine was weaker and less voltage dependent (Kd(0) = 37·8 ìÒ and zä = 0·55); ä and è are electrical distances measured from the outside and inside, respectively. 6. 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 zä values estimated for block by internal spermine predicted much stronger external block than was measured experimentally, and vice versa. 7. An Eyring rate theory model with two energy wells and three barriers explained qualitatively many characteristic features of the action of polyamines on GluRs, including biphasic I-V relationships, weaker block by external than internal spermine and low permeability.

Q/R Site Editing Controls Kainate Receptor Inhibition by Membrane Fatty Acids

“…In contrast to the potent block of unedited channels by internal and external polyamines (Bowie and Mayer, 1995;Kamboj et al, 1995;Bähring et al, 1997;Mott et al, 2003), we find that extracellular fatty acid applications only produce strong inhibition of recombinant kainate receptor channels that are composed of fully edited subunits. Heteromeric GluR5(Q)/GluR6(R) or GluR5(R)/GluR6(Q) channels were inhibited much less than were fully edited GluR5(R)/GluR6(R) heteromeric receptors or homomeric GluR6(R) receptors.…”

“…Channel block by intracellular and extracellular polyamines, which preferentially affect channels lacking an R within the pore, displays significant voltage dependence (Bowie and Mayer, 1995;Kamboj et al, 1995), although dependence on voltage is weaker for inhibition by extracellular versus intracellular polyamines (Bähring et al, 1997). If penetration of the negatively charged fatty acid carboxyl group into the channel pore was required for interaction with the positively charged side chains of arginine at the Q/R site, potency of block would be expected to change as the voltage gradient along the axis of the pore varied.…”

“…Channels composed only of subunits that contain a Q at this site are permeable to sodium, potassium, and calcium and are subject to voltagedependent block by internal polyamines, which produces strong inward rectification of the current-voltage relationship (Dingledine et al, 1999). In contrast, the inclusion of one or more subunits that contain an R at the Q/R site reduces the single-channel conductance (Howe, 1996;Swanson et al, 1996), eliminates block by polyamines (Bowie and Mayer, 1995;Kamboj et al, 1995;Bähring et al, 1997;Mott et al, 2003), and reduces channel permeability to calcium (Egebjerg and Heinemann, 1993;Köhler et al, 1993;Burnashev et al, 1995Burnashev et al, , 1996.…”

Presynaptic Kainate Receptor Activation Is a Novel Mechanism for Target Cell-Specific Short-Term Facilitation at Schaffer Collateral Synapses

“…Patch electrodes (3-4.5 M⍀) were filled with internal solution composed of the following (in mM): 100 Cs-Gluconate, 0.6 EGTA, 5.0 MgCl 2 , 10 HEPES, pH was adjusted to 7.2 with CsOH. The internal solution also contained 10 mM BAPTA to block interneuron LTP and LTD (Laezza et al, 1999), and to inhibit postsynaptic Ca 2ϩ -mediated effects of G-protein coupled receptors and prevent postsynaptic short-term plasticity; triethylammonium chloride] (5 mM) to improve space clamp and reduce nonlinear effects caused by voltagegated channels in dendrites while recording from the soma; 10 mM ATP to chelate intracellular polyamines and prevent possible postsynaptic short-term plasticity at calcium-permeable AMPA receptors (Bahring et al, 1997;McBain, 1998;Rozov and Burnashev, 1999;Toth et al, 2000). The access resistance and holding current (Ͻ200 pA) were monitored continuously.…”

Functional reconstitution ofDrosophila melanogasterNMJ glutamate receptors

“…S3). It is well established that such biphasic rectification is produced by voltage-dependent ion channel block by endogenous cytoplasmic spermine and spermidine, with polyamine permeation on depolarization to positive membrane potentials (28,36). Analysis of conductance voltage plots revealed a 13-mV depolarizing shift in the half-block potential for GluRIIB/C/D/E −31.9 ± 1.3 mV (n = 6) compared with GluRIIA/C/D/E −44.8 ± 0.9 mV (n = 9), suggesting a lower polyamine affinity for channels containing the GluRIIB subunit (Fig.…”