Stable Open State Research Articles

Gating Reaction Mechanisms for NMDA Receptor Channels

NMDA receptors (NMDARs) mediate the slow component of excitatory transmission in the CNS and play key roles in synaptic plasticity and excitotoxicity. We investigated the gating reaction mechanism of fully liganded NR1/NR2A recombinant NMDARs (expressed in Xenopus oocytes) by fitting all possible three-closed/two-open-state, noncyclic kinetic schemes to currents elicited by saturating concentrations of glutamate plus glycine. The adequacy of each scheme was assessed by maximum likelihood values and autocorrelation coefficients of single-channel currents, as well as by the predicted time courses of transient macroscopic currents. Two schemes provided the best description for NMDAR gating at both the single-channel and macroscopic levels. These two schemes had coupled open states, only one gateway between the closed and open aggregates, and at least two preopening closed states. These two models could be condensed into a cyclic reaction mechanism. Using a linear reaction scheme, the overall "gating" rates (from the initial stable closed state to the final stable open state) are 177 and 4.4 s(-1).

The Link between Ion Permeation and Inactivation Gating of Kv4 Potassium Channels

Kv4 potassium channels undergo rapid inactivation but do not seem to exhibit the classical N-type and C-type mechanisms present in other Kv channels. We have previously hypothesized that Kv4 channels preferentially inactivate from the preopen closed state, which involves regions of the channel that contribute to the internal vestibule of the pore. To further test this hypothesis, we have examined the effects of permeant ions on gating of three Kv4 channels (Kv4.1, Kv4.2, and Kv4.3) expressed in Xenopus oocytes. Rb + is an excellent tool for this purpose because its prolonged residency time in the pore delays K + channel closing. The data showed that, only when Rb + carried the current, both channel closing and the development of macroscopic inactivation are slowed (1.5- to 4-fold, relative to the K + current). Furthermore, macroscopic Rb + currents were larger than K + currents (1.2- to 3-fold) as the result of a more stable open state, which increases the maximum open probability. These results demonstrate that pore occupancy can influence inactivation gating in a manner that depends on how channel closing impacts inactivation from the preopen closed state. By examining possible changes in ionic selectivity and the influence of elevating the external K + concentration, additional experiments did not support the presence of C-type inactivation in Kv4 channels.

A Single Transmembrane Site in the KCNE-encoded Proteins Controls the Specificity of KvLQT1 Channel Gating

KCNEs are a family of genes encoding small integral membrane proteins whose role in governing voltage-gated potassium channel gating is emerging. Whether each member of this homologous family interacts with channel proteins in the same manner is unknown; however, it is clear that the functional effect of each KCNE on channel gating is different. The specificity of KCNE1 (minK) and KCNE3 control of activation of the potassium channel KvLQT1 maps to a triplet of amino acids within the KCNE transmembrane domain by chimera analysis. We now define the structural determinants of functional specificity within this triplet. The central amino acid of the triplet (Thr-58 of minK and Val-72 of KCNE3) is essential for the specific control of voltage-dependent channel activation characteristics of both minK and KCNE3. Using site-directed mutations that substitute minK and KCNE3 residues, we determined that a hydroxylated central amino acid is necessary for the slow sigmoidal activation produced by minK. The precise spacing of the hydroxyl group was required for minK-like activation. An aliphatic amino acid substituted at position 58 of minK is capable of reproducing KCNE3-like kinetics and voltage-independent constitutive current activation. The bulk of the central residue is another critical parameter, indicating precise positioning of this portion of the KCNE proteins within the channel complex. An intermediate phenotype produced by several smaller aliphatic-substituted mutants yields conditional voltage independence that is distinct from the voltage-dependent gating process, suggesting that KCNE3 traps the channel in a stable open state. From these results, we propose a model of KCNE-potassium channel interaction where the functional consequence depends on the precise contact at a single amino acid.

Inactivation gating of Kv4 potassium channels: molecular interactions involving the inner vestibule of the pore.

Kv4 channels represent the main class of brain A-type K+ channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174- 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J. 72:163-174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603-626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316-2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance-voltage curve ( approximately 5 mV) and the prepulse inactivation curve (>10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K+ channel that undergoes N- and C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4-specific residue in the S4-S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404, 406]I. However, this mutant did not exhibit disrupted closed state inactivation. A kinetic model that assumes coupling between channel closing and inactivation at depolarized membrane potentials accounts for the results. We propose that components of the pore's internal vestibule control both closing and inactivation in Kv4 K+ channels.

Single channel seeks permeant ion for brief but intimate relationship.

Single channel seeks permeant ion for brief but intimate relationship.