Hans-Günther Knaus

Presynaptic Ca2+-Activated K+ Channels in Glutamatergic Hippocampal Terminals and Their Role in Spike Repolarization and Regulation of Transmitter Release

Large-conductance Ca2+-activated K+ channels (BK, also called Maxi-K orSlo channels) are widespread in the vertebrate nervous system, but their functional roles in synaptic transmission in the mammalian brain are largely unknown. By combining electrophysiology and immunogold cytochemistry, we demonstrate the existence of functional BK channels in presynaptic terminals in the hippocampus and compare their functional roles in somata and terminals of CA3 pyramidal cells. Double-labeling immunogold analysis with BK channel and glutamate receptor antibodies indicated that BK channels are targeted to the presynaptic membrane facing the synaptic cleft in terminals of Schaffer collaterals in stratum radiatum. Whole-cell, intracellular, and field-potential recordings from CA1 pyramidal cells showed that the presynaptic BK channels are activated by calcium influx and can contribute to repolarization of the presynaptic action potential (AP) and negative feedback control of Ca2+ influx and transmitter release. This was observed in the presence of 4-aminopyridine (4-AP, 40–100 μm), which broadened the presynaptic compound action potential. In contrast, the presynaptic BK channels did not contribute significantly to regulation of action potentials or transmitter release under basal experimental conditions, i.e., without 4-AP, even at high stimulation frequencies. This is unlike the situation in the parent cell bodies (CA3 pyramidal cells), where BK channels contribute strongly to action potential repolarization. These results indicate that the functional role of BK channels depends on their subcellular localization.

High-conductance calcium-activated potassium channels; structure, pharmacology, and function.

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K+ channels. They are unique in their dual requirement for depolarization and Ca2+ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, α and Β. The α subunit is a member of theslo Ca2+-activated K+ channel gene family and forms the ion conduction pore. The Β subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against α and Β subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.

The Epilepsy-Linked Lgi1 Protein Assembles into Presynaptic Kv1 Channels and Inhibits Inactivation by Kvβ1

The voltage-gated potassium (Kv) channel subunit Kv1.1 is a major constituent of presynaptic A-type channels that modulate synaptic transmission in CNS neurons. Here, we show that Kv1.1-containing channels are complexed with Lgi1, the functionally unassigned product of the leucine-rich glioma inactivated gene 1 (LGI1), which is causative for an autosomal dominant form of lateral temporal lobe epilepsy (ADLTE). In the hippocampal formation, both Kv1.1 and Lgi1 are coassembled with Kv1.4 and Kvbeta1 in axonal terminals. In A-type channels composed of these subunits, Lgi1 selectively prevents N-type inactivation mediated by the Kvbeta1 subunit. In contrast, defective Lgi1 molecules identified in ADLTE patients fail to exert this effect resulting in channels with rapid inactivation kinetics. The results establish Lgi1 as a novel subunit of Kv1.1-associated protein complexes and suggest that changes in inactivation gating of presynaptic A-type channels may promote epileptic activity.

Quantitative proteomics of the Cav2 channel nano-environments in the mammalian brain

Local Ca2+ signaling occurring within nanometers of voltage-gated Ca2+ (Cav) channels is crucial for CNS function, yet the molecular composition of Cav channel nano-environments is largely unresolved. Here, we used a proteomic strategy combining knockout-controlled multiepitope affinity purifications with high-resolution quantitative MS for comprehensive analysis of the molecular nano-environments of the Cav2 channel family in the whole rodent brain. The analysis shows that Cav2 channels, composed of pore-forming α1 and auxiliary β subunits, are embedded into protein networks that may be assembled from a pool of ∼200 proteins with distinct abundance, stability of assembly, and preference for the three Cav2 subtypes. The majority of these proteins have not previously been linked to Cav channels; about two-thirds are dedicated to the control of intracellular Ca2+ concentration, including G protein-coupled receptor-mediated signaling, to activity-dependent cytoskeleton remodeling or Ca2+-dependent effector systems that comprise a high portion of the priming and release machinery of synaptic vesicles. The identified protein networks reflect the cellular processes that can be initiated by Cav2 channel activity and define the molecular framework for organization and operation of local Ca2+ signaling by Cav2 channels in the brain.

K+ channel expression during B cell differentiation: implications for immunomodulation and autoimmunity.

Using whole-cell patch-clamp, fluorescence microscopy and flow cytometry, we demonstrate a switch in potassium channel expression during differentiation of human B cells from naive to memory cells. Naive and IgD+CD27+ memory B cells express small numbers of the voltage-gated Kv1.3 and the Ca2+-activated intermediate-conductance IKCa1 channel when quiescent, and increase IKCa1 expression 45-fold upon activation with no change in Kv1.3 levels. In contrast, quiescent class-switched memory B cells express high levels of Kv1.3 (∼2000 channels/cell) and maintain their Kv1.3high expression after activation. Consistent with their channel phenotypes, proliferation of naive and IgD+CD27+ memory B cells is suppressed by the specific IKCa1 inhibitor TRAM-34 but not by the potent Kv1.3 blocker Stichodactyla helianthus toxin, whereas the proliferation of class-switched memory B cells is suppressed by Stichodactyla helianthus toxin but not TRAM-34. These changes parallel those reported for T cells. Therefore, specific Kv1.3 and IKCa1 inhibitors may have use in therapeutic manipulation of selective lymphocyte subsets in immunological disorders.

Comparative immunohistochemical distribution of three small-conductance Ca2+-activated potassium channel subunits, SK1, SK2, and SK3 in mouse brain

To investigate the distribution of all three SK channel subunits in the mouse central nervous system, we performed immunohistochemistry using sequence-specific antibodies directed against SK1, SK2, and SK3 proteins. Expression of SK1 and SK2 proteins revealed a partly overlapping distribution pattern restricted to a limited number of brain areas (e.g., neocortex, hippocampal formation). In contrast, SK3 immunoreactivity was rather complementary and predominantly detected in phylogenetically older brain regions like basal ganglia, thalamus, and various brain stem nuclei (e.g., locus coeruleus, tegmental nuclei). At the cellular level, SK1- and SK2-like immunoreactivity was primarily localized to somatic and dendritic structures, whereas the majority of SK3-like immunoreactivity was associated with varicose fibers.

PHARMACOLOGY OF POTASSIUM CHANNELS

Publisher Summary Potassium channels represent the largest and most diverse family of ion channels. K + channels can be divided into two groups, voltage-gated and ligand-gated channels, depending on the stimulus that triggers the conformational changes leading to channel opening. K + channels share in common the feature of having high selectivity for K + as the permeating ion. Because of this property, and given the wide tissue distribution of these proteins, K + channels have been postulated to be involved in a variety of physiologic processes, such as control of cell excitability, release of neurotransmitters, secretion of hormones, regulation of fluid secretion, and clonal expansion of cells of the immune system. This chapter mentions the nature and properties of the specific channels those are present. A large number of voltage-dependent K + channels are known to exist. They are presumed to contain six α-helical transmembrane domains (S1-S6) with a segment between S5 and S6, termed the P region that contributes to the channels pore. The P region is the most conserved domain among all different types of K + channels and, because it is not large enough to cross the membrane in an α-helical conformation, it has been proposed to form a p-hairpin-like structure. Given the fact that some peptidyl blockers display a broad spectrum of interaction with different family members, this review is divided into three major areas; voltage-gated K + channels, Ca 2+ - activated K + channels, and ATP-dependent K + channels. Discussed are the peptidyl blockers derived and the peptidyl inhibitors isolated from scorpion venoms, the peptidyl blockers from sea anemone and the spider venom and nonpeptidyl blockers. Ca 2+ -activated K + channels are discussed; including the interaction of the peptide ChTX with maxi-K channels and the nonpeptidyl maxi-K channel modulators. Also discussed are the small-conductance Ca 2+ -activated K + channels and the ATP-dependent K + channels.

Ca2+ -activated K+ channels of the BK-type in the mouse brain.

An antibody against the 442 carboxy-terminal amino acids of the BK channel α-subunit detects high immunoreactivity within the telencephalon in cerebral cortices, olfactory bulb, basal ganglia and hippocampus, while lower levels are found in basal forebrain regions and amygdala. Within the diencephalon, high density was found in nuclei of the ventral and dorsal thalamus and the medial habenular nucleus, and low density in the hypothalamus. The fasciculus retroflexus and its termination in the mesencephalic interpeduncular nucleus are prominently stained. Other mesencephalic expression sites are periaquaeductal gray and raphe nuclei. In the rhombencephalon, BK channels are enriched in the cerebellar cortex and in the locus coeruleus. Strong immunoreactivity is also contained in the vestibular nuclei, but not in cranial nerves and their intramedullary course of their roots. On the cellular level, BK channels show pre- and postsynaptic localizations, i.e., in somata, dendrites, axons and synaptic terminals.

Complex Subunit Assembly of Neuronal Voltage-gated K+Channels BASIS FOR HIGH-AFFINITY TOXIN INTERACTIONS AND PHARMACOLOGY

Neurons require specific patterns of K+ channel subunit expression as well as the precise coassembly of channel subunits into heterotetrameric structures for proper integration and transmission of electrical signals. In vivo subunit coassembly was investigated by studying the pharmacological profile, distribution, and subunit composition of voltage-gated Shaker family K+(Kv1) channels in rat cerebellum that are labeled by125I-margatoxin (125I-MgTX;K d , 0.08 pm). High-resolution receptor autoradiography showed spatial receptor expression mainly in basket cell terminals (52% of all cerebellar sites) and the molecular layer (39% of sites). Sequence-directed antibodies indicated overlapping expression of Kv1.1 and Kv1.2 in basket cell terminals, whereas the molecular layer expressed Kv1.1, Kv1.2, Kv1.3, and Kv1.6 proteins. Immunoprecipitation experiments revealed that all 125I-MgTX receptors contain at least one Kv1.2 subunit and that 83% of these receptors are heterotetramers of Kv1.1 and Kv1.2 subunits. Moreover, 33% of these Kv1.1/Kv1.2-containing receptors possess either an additional Kv1.3 or Kv1.6 subunit. Only a minority of the 125I-MgTX receptors (<20%) seem to be homotetrameric Kv1.2 channels. Heterologous coexpression of Kv1.1 and Kv1.2 subunits in COS-1 cells leads to the formation of a complex that combines the pharmacological profile of both parent subunits, reconstituting the native MgTX receptor phenotype. Subunit assembly provides the structural basis for toxin binding pharmacology and can lead to the association of as many as three distinct channel subunits to form functional K+channels in vivo.

Photoaffinity labelling of the phenylalkylamine receptor of the skeletal muscle transverse-tubule calcium channel

The tritiated arylazido phenylalkylamine (‐)‐5‐[(3‐azidophenethyl)[N‐methyl‐3H]methylamino]‐2‐(3,4,5‐tri‐methoxyphenyl)‐2‐isopropylvaleronitrile was synthesized and used to photoaffinity label the phenylalkylamine receptor of the membrane‐bound and purified calcium channel from guinea‐pig skeletal muscle trans‐verse‐tubule membranes. The photoaffinity ligand binds reversibly to partially purified membranes with a K d of 2.0 ± 0.5 nM and a B max of 17.0 ± 0.9 protein. Binding is stereospecifically regulated by all three classes of organic calcium channel drugs. A 155 kDa band was specifically photolabelled in transverse‐tubule particulate and purified calcium channel preparations after ultraviolet irradiation. Additional minor labelled polypeptides (92, 60 and 33 kDa) were only observed in membranes. The heterogeneous 155 kDa region of the purified channel was resolved into two distinct silver‐stained polypeptides after reduction (i.e. 155 and 135 kDa). Only the 155 kDa polypeptide carries the photoaffinity label and it is concluded that the 135 kDa polypeptide (which migrates as a 165 kDa band under alkylating conditions) is not a high‐affinity drug receptor carrying subunit of the skeletal muscle transverse‐tubule L‐type calcium channel.