Excitability. Excitability of cell membranes is crucial for signaling in many types of cell. Excitation in the physiological sense means that the cell membrane potential undergoes characteristic changes which, in most cases, go in the depolarizing direction. Single depolarization from the resting potential to potentials near 0 mV has generally been called an action potential. A schematic representation of a neuronal action potential is given in Fig. 12.1 A. The action potential is triggered when the membrane potential, which was at the resting level, depolarizes and reaches the threshold of excitation. This depolarization, which triggers the action potential, is generated by depolarizing synaptic currents, or depolarizing current coming from a membrane region that is already excited (propagation of an action potential), or by pacemaker currents mediated by pacemaker channels, or by current injected externally by an electrode. The duration of different types of action potential varies from seconds to less than 1 ms.

Ion Channels in Excitable Membranes

Voltage-dependent potassium ion (K+) channels (Kv channels) conduct K+ ions across the cell membrane in response to changes in the membrane voltage, thereby regulating neuronal excitability by modulating the shape and frequency of action potentials. Here we report the crystal structure, at a resolution of 2.9 angstroms, of a mammalian Kv channel, Kv1.2, which is a member of the Shaker K+ channel family. This structure is in complex with an oxido-reductase beta subunit of the kind that can regulate mammalian Kv channels in their native cell environment. The activation gate of the pore is open. Large side portals communicate between the pore and the cytoplasm. Electrostatic properties of the side portals and positions of the T1 domain and beta subunit are consistent with electrophysiological studies of inactivation gating and with the possibility of K+ channel regulation by the beta subunit.

http://science.sciencemag.org/content/sci/309/5736/897.full.pdf

Crystal Structure of a Mammalian Voltage-Dependent Shaker Family K + Channel

This chapter discusses the gamma-aminobutyric acid (GABA) receptor channels, which are the most abundant inhibitory neurotransmitter in the CNS. Following release from presynaptic vesicles, GABA exerts fast inhibitory effects by interacting with GABA receptors, whose primary function is to hyperpolarize neuronal membranes in mature CNS neurons. GABA receptors are found both presynaptically, where they decrease the likelihood of neurotransmitter release, and postsynaptically, where they decrease the likelihood of neuronal firing. There are two types of GABA receptor, termed GABA A and GABA B receptors. GABA A receptors are fast-activating Clˉ channels from the Cys-loop family of ligand-gated ion channels. Activation of GABA A receptors causes membrane hyperpolarization by allowing Clˉ influx, reflecting the relatively low concentration of Clˉ found intracellularly in most adult CNS neurons. GABA A receptors can also mediate depolarizing responses in most immature CNS neurons and in mature peripheral neurons.

GABAA Receptor Channels

Ion transport proteins must remove an ion's hydration shell to coordinate the ion selectively on the basis of its size and charge. To discover how the K+ channel solves this fundamental aspect of ion conduction, we solved the structure of the KcsA K+ channel in complex with a monoclonal Fab antibody fragment at 2.0 A resolution. Here we show how the K+ channel displaces water molecules around an ion at its extracellular entryway, and how it holds a K+ ion in a square antiprism of water molecules in a cavity near its intracellular entryway. Carbonyl oxygen atoms within the selectivity filter form a very similar square antiprism around each K+ binding site, as if to mimic the waters of hydration. The selectivity filter changes its ion coordination structure in low K+ solutions. This structural change is crucial to the operation of the selectivity filter in the cellular context, where the K+ ion concentration near the selectivity filter varies in response to channel gating.

Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution.

The potassium channels encoded by the Drosophila Shaker gene activate and inactivate rapidly when the membrane potential becomes more positive. Site-directed mutagenesis and single-channel patch-clamp recording were used to explore the molecular transitions that underlie inactivation in Shaker potassium channels expressed in Xenopus oocytes. A region near the amino terminus with an important role in inactivation has now been identified. The results suggest a model where this region forms a cytoplasmic domain that interacts with the open channel to cause inactivation.

https://science.sciencemag.org/content/sci/250/4980/533.full.pdf

Biophysical and molecular mechanisms of shaker potassium channel inactivation

Site-directed mutagenesis experiments have suggested a model for the inactivation mechanism of Shaker potassium channels from Drosophila melanogaster. In this model, the first 20 amino acids form a cytoplasmic domain that interacts with the open channel to cause inactivation. The model was tested by the internal application of a synthetic peptide, with the sequence of the first 20 residues of the ShB alternatively spliced variant, to noninactivating mutant channels expressed in Xenopus oocytes. The peptide restored inactivation in a concentration-dependent manner. Like normal inactivation, peptide-induced inactivation was not noticeably voltage-dependent. Trypsin-treated peptide and peptides with sequences derived from the first 20 residues of noninactivating mutants did not restore inactivation. These results support the proposal that inactivation occurs by a cytoplasmic domain that occludes the ion-conducting pore of the channel.

https://science.sciencemag.org/content/sci/250/4980/568.full.pdf

Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB

Two types of inactivation in Shaker K+ channels: Effects of alterations in the carboxy-terminal region

Predictions of different classes of gating models involving identical conformational changes in each of four subunits were compared to the gating behavior of Shaker potassium channels without N-type inactivation. Each model was tested to see if it could simulate the voltage dependence of the steady state open probability, and the kinetics of the single-channel currents, macroscopic ionic currents and macroscopic gating currents using a single set of parameters. Activation schemes based upon four identical single-step activation processes were found to be incompatible with the experimental results, as were those involving a concerted, opening transition. A model where the opening of the channel requires two conformational changes in each of the four subunits can adequately account for the steady state and kinetic behavior of the channel. In this model, the gating in each subunit is independent except for a stabilization of the open state when all four subunits are activated, and an unstable closed conformation that the channel enters after opening. A small amount of negative cooperativity between the subunits must be added to account quantitatively for the dependence of the activation time course on holding voltage.

https://rupress.org/jgp/article-pdf/103/2/321/1185823/321.pdf

Shaker potassium channel gating. III: Evaluation of kinetic models for activation

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.

/pdf/shaker-potassium-channel-gating-ii-transitions-in-the-3eflekcy6p.pdf

William N. Zagotta

Papers

Biophysical and molecular mechanisms of shaker potassium channel inactivation

Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB

Two types of inactivation in Shaker K+ channels: Effects of alterations in the carboxy-terminal region

Shaker potassium channel gating. III: Evaluation of kinetic models for activation

Shaker potassium channel gating. II: Transitions in the activation pathway.