Light-regulated voltage-gated potassium channels for acute interrogation of channel function in neurons and behavior.

Voltage-gated potassium (Kv) channels regulate the membrane potential and conductance of excitable cells to control the firing rate and waveform of action potentials. Even though Kv channels have been intensely studied for over 70 year, surprisingly little is known about how specific channels expressed in various neurons and their functional properties impact neuronal network activity and behavior in vivo. Although many in vivo genetic manipulations of ion channels have been tried, interpretation of these results is complicated by powerful homeostatic plasticity mechanisms that act to maintain function following perturbations in excitability.

Modulatory mechanisms and multiple functions of somatodendritic A-type K (+) channel auxiliary subunits.

Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs).

S-glutathionylation of an auxiliary subunit confers redox sensitivity to Kv4 channel inactivation.

Reactive oxygen species (ROS) regulate ion channels, modulate neuronal excitability, and contribute to the etiology of neurodegenerative disorders. ROS differentially suppress fast "ball-and-chain" N-type inactivation of cloned Kv1 and Kv3 potassium channels but not of Kv4 channels, likely due to a lack of reactive cysteines in Kv4 N-termini. Recently, we discovered that N-type inactivation of Kv4 channel complexes can be independently conferred by certain N-terminal variants of Kv4 auxiliary subunits (DPP6a, DPP10a).

A conserved pre-block interaction motif regulates potassium channel activation and N-type inactivation.

N-type inactivation occurs when the N-terminus of a potassium channel binds into the open pore of the channel. This study examined the relationship between activation and steady state inactivation for mutations affecting the N-type inactivation properties of the Aplysia potassium channel AKv1 expressed in Xenopus oocytes. The results show that the traditional single-step model for N-type inactivation fails to properly account for the observed relationship between steady state channel activation and inactivation curves.

Functional stoichiometry underlying KChIP regulation of Kv4.2 functional expression.

K channel-interacting proteins (KChIPs) enhance functional expression of Kv4 channels by binding to an N-terminal regulatory region located in the first 40 amino acids of Kv4.2 that we call the functional expression regulating N-terminal (FERN) domain. Mutating two residues in the FERN domain to alanines, W8A and F11A, disrupts KChIP binding and regulation of Kv4.

Conserved N-terminal negative charges support optimally efficient N-type inactivation of Kv1 channels.

N-type inactivation is produced by the binding of a potassium channel's N-terminus within the open pore, blocking conductance. Previous studies have found that introduction of negative charges into N-terminal inactivation domains disrupts inactivation; however, the Aplysia AKv1 N-type inactivation domain contains two negatively charged residues, E2 and E9. Rather than being unusual, sequence analysis shows that this N-terminal motif is highly conserved among Kv1 sequences across many phyla.

A new TASK for Dipeptidyl Peptidase-like Protein 6.

Dipeptidyl Peptidase-like Protein 6 (DPP6) is widely expressed in the brain where it co-assembles with Kv4 channels and KChIP auxiliary subunits to regulate the amplitude and functional properties of the somatodendritic A-current, ISA. Here we show that in cerebellar granule (CG) cells DPP6 also regulates resting membrane potential and input resistance by increasing the amplitude of the IK(SO) resting membrane current. Pharmacological analysis shows that DPP6 acts through the control of a channel with properties matching the K2P channel TASK-3.

Incorporation of DPP6a and DPP6K variants in ternary Kv4 channel complex reconstitutes properties of A-type K current in rat cerebellar granule cells.

Dipeptidyl peptidase-like protein 6 (DPP6) proteins co-assemble with Kv4 channel α-subunits and Kv channel-interacting proteins (KChIPs) to form channel protein complexes underlying neuronal somatodendritic A-type potassium current (I(SA)). DPP6 proteins are expressed as N-terminal variants (DPP6a, DPP6K, DPP6S, DPP6L) that result from alternative mRNA initiation and exhibit overlapping expression patterns. Here, we study the role DPP6 variants play in shaping the functional properties of I(SA) found in cerebellar granule (CG) cells using quantitative RT-PCR and voltage-clamp recordings of whole-cell currents from reconstituted channel complexes and native I(SA) channels.

Dipeptidyl peptidase-like protein 6 is required for normal electrophysiological properties of cerebellar granule cells.

In cerebellar granule (CG) cells and many other neurons, A-type potassium currents play an important role in regulating neuronal excitability, firing patterns, and activity-dependent plasticity. Protein biochemistry has identified dipeptidyl peptidase-like protein 6 (DPP6) as an auxiliary subunit of Kv4-based A-type channels and thus a potentially important regulator of neuronal excitability. In this study, we used an RNA interference (RNAi) strategy to examine the role DPP6 plays in forming and shaping the electrophysiological properties of CG cells.

A novel N-terminal motif of dipeptidyl peptidase-like proteins produces rapid inactivation of KV4.2 channels by a pore-blocking mechanism.

The somatodendritic subthreshold A-type K(+) current in neurons (I(SA)) depends on its kinetic and voltage-dependent properties to regulate membrane excitability, action potential repetitive firing, and signal integration. Key functional properties of the K(V)4 channel complex underlying I(SA) are determined by dipeptidyl peptidase-like proteins known as dipeptidyl peptidase 6 (DPP6) and dipeptidyl peptidase 10 (DPP10). Among the multiple known DPP10 isoforms with alternative N-terminal sequences, DPP10a confers exceptionally fast inactivation to K(V)4.

Multiple intermediate states precede pore block during N-type inactivation of a voltage-gated potassium channel.

N-type inactivation of voltage-gated potassium channels is an autoinhibitory process that occurs when the N terminus binds within the channel pore and blocks conduction. N-type inactivation and recovery occur with single-exponential kinetics, consistent with a single-step reaction where binding and block occur simultaneously. However, recent structure-function studies have suggested the presence of a preinactivated state whose formation and loss regulate inactivation and recovery kinetics.

Multiple Kv channel-interacting proteins contain an N-terminal transmembrane domain that regulates Kv4 channel trafficking and gating.

Kv channel-interacting proteins (KChIPs) are auxiliary subunits of the heteromultimeric channel complexes that underlie neuronal I(SA), the subthreshold transient K(+) current that dynamically regulates membrane excitability, action potential firing properties, and long term potentiation. KChIPs form cytoplasmic associations with the principal pore-forming Kv4 subunits and typically mediate enhanced surface expression and accelerated recovery from depolarization-induced inactivation. An exception is KChIP4a, which dramatically suppresses Kv4 inactivation while promoting neither surface expression nor recovery.

DPP10 splice variants are localized in distinct neuronal populations and act to differentially regulate the inactivation properties of Kv4-based ion channels.

Dipeptidyl peptidase-like proteins (DPLs) and Kv-channel-interacting proteins (KChIPs) join Kv4 pore-forming subunits to form multi-protein complexes that underlie subthreshold A-type currents (I(SA)) in neuronal somatodendritic compartments. Here, we characterize the functional effects and brain distributions of N-terminal variants belonging to the DPL dipeptidyl peptidase 10 (DPP10). In the Kv4.

Zn2+-dependent redox switch in the intracellular T1-T1 interface of a Kv channel.

The thiol-based redox regulation of proteins plays a central role in cellular signaling. Here, we investigated the redox regulation at the Zn(2+) binding site (HX(5)CX(20)CC) in the intracellular T1-T1 inter-subunit interface of a Kv4 channel. This site undergoes conformational changes coupled to voltage-dependent gating, which may be sensitive to oxidative stress.

Manipulating Kv4.2 identifies a specific component of hippocampal pyramidal neuron A-current that depends upon Kv4.2 expression.

The somatodendritic A-current, I(SA), in hippocampal CA1 pyramidal neurons regulates the processing of synaptic inputs and the amplitude of back propagating action potentials into the dendritic tree, as well as the action potential firing properties at the soma. In this study, we have used RNA interference and over-expression to show that expression of the Kv4.2 gene specifically regulates the I(SA) component of A-current in these neurons.

Regulation of surface localization of the small conductance Ca2+-activated potassium channel, Sk2, through direct phosphorylation by cAMP-dependent protein kinase.

Small conductance, Ca2+-activated voltage-independent potassium channels (SK channels) are widely expressed in diverse tissues; however, little is known about the molecular regulation of SK channel subunits. Direct alteration of ion channel subunits by kinases is a candidate mechanism for functional modulation of these channels. We find that activation of cyclic AMP-dependent protein kinase (PKA) with forskolin (50 microm) causes a dramatic decrease in surface localization of the SK2 channel subunit expressed in COS7 cells due to direct phosphorylation of the SK2 channel subunit.

NMR-derived dynamic aspects of N-type inactivation of a Kv channel suggest a transient interaction with the T1 domain.

Some eukaryotic voltage-gated K+ (Kv) channels contain an N-terminal inactivation peptide (IP), which mediates a fast inactivation process that limits channel function during membrane depolarization and thus shapes the action potential. We obtained sequence-specific nuclear magnetic resonance (NMR) assignments for the polypeptide backbone of a tetrameric N-terminal fragment (amino acids 1-181) of the Aplysia Kv1.1 channel.

Multiprotein assembly of Kv4.2, KChIP3 and DPP10 produces ternary channel complexes with ISA-like properties.

Kv4 pore-forming subunits are the principal constituents of the voltage-gated K+ channel underlying somatodendritic subthreshold A-type currents (I(SA)) in neurones. Two structurally distinct types of Kv4 channel modulators, Kv channel-interacting proteins (KChIPs) and dipeptidyl-peptidase-like proteins (DPLs: DPP6 or DPPX, DPP10 or DPPY), enhance surface expression and modify functional properties. Since KChIP and DPL distributions overlap in the brain, we investigated the potential coassembly of Kv4.

Functionally active t1-t1 interfaces revealed by the accessibility of intracellular thiolate groups in kv4 channels.

Gating of voltage-dependent K(+) channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents.

Molecular physiology and modulation of somatodendritic A-type potassium channels.

The somatodendritic subthreshold A-type K+ current (ISA) in nerve cells is a critical component of the ensemble of voltage-gated ionic currents that determine somatodendritic signal integration. The underlying K+ channel belongs to the Shal subfamily of voltage-gated K+ channels. Most Shal channels across the animal kingdom share a high degree of structural conservation, operate in the subthreshold range of membrane potentials, and exhibit relatively fast inactivation and recovery from inactivation.

KChIP3 rescues the functional expression of Shal channel tetramerization mutants.

KChIP proteins regulate Shal, Kv4.x, channel expression by binding to a conserved sequence at the N terminus of the subunit. The binding of KChIP facilitates a redistribution of Kv4 protein to the cell surface, producing a large increase in current along with significant changes in channel gating kinetics.

Modulation of Kv4.2 channel expression and gating by dipeptidyl peptidase 10 (DPP10).

The dipeptidyl aminopeptidase-like protein DPPX (DPP6) associates with Kv4 potassium channels, increasing surface trafficking and reconstituting native neuronal ISA-like properties. Dipeptidyl peptidase 10 (DPP10) shares with DPP6 a high amino acid identity, lack of enzymatic activity, and expression predominantly in the brain. We used a two-electrode voltage-clamp and oocyte expression system to determine if DPP10 also interacts with Kv4 channels and modulates their expression and function.

Structural insights into the functional interaction of KChIP1 with Shal-type K(+) channels.

Four Kv channel-interacting proteins (KChIP1 through KChIP4) interact directly with the N-terminal domain of three Shal-type voltage-gated potassium channels (Kv4.1, Kv4.2, and Kv4.

Determining the basis of channel-tetramerization specificity by x-ray crystallography and a sequence-comparison algorithm: Family Values (FamVal).

We have developed a semiempirical algorithm called Family Values (FamVal), which identifies residues that encode functional specificity in a protein sequence. Given a multiple sequence alignment (MSA) grouped into functionally distinct subfamilies, FamVal calculates a specificity score for each subfamily at every amino acid position of an MSA. This algorithm was used to predict specificity-encoding positions within the tetramerization assembly (T1) domain of voltage-gated potassium (Kv) channel subfamilies Kv3 and Kv4.

The role of Zn2+ in Shal voltage-gated potassium channel formation.

Voltage-gated potassium channels are formed by the tetramerization of their alpha subunits, in a process that is controlled by their conserved N-terminal T1 domains. The crystal structures of Shaker and Shaw T1 domains reveal interesting differences in structures that are contained within a highly conserved BTB/POZ domain fold. The most surprising difference is that the Shaw T1 domain contains an intersubunit Zn2+ ion that is lacking in the Shaker T1 domain.