Biomedical subjects

Bernd Fakler

Publications and source records attributed to Bernd Fakler.

13 recordsLinked to original sources

PubMed2006

The epilepsy-linked Lgi1 protein assembles into presynaptic Kv1 channels and inhibits inactivation by Kvbeta1.

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.

Amino Acid Sequence↗

PubMed2005

Ca2+-independent activation of BKCa channels at negative potentials in mammalian inner hair cells.

The defining characteristic of large-conductance Ca(2)(+)- and voltage-activated K(+) channels (BK(Ca)) is their allosteric activation by two distinct stimuli, membrane depolarization and cytosolic Ca(2)(+) ions. In this allosteric gating, increasing cytosolic Ca(2)(+) concentration ([Ca(2)(+)](i)) shifts the depolarization required for channel opening into the physiological voltage range. In fact, according to present knowledge, elevation of [Ca(2)(+)](i) to micromolar levels is the only means to activate BK(Ca) at membrane potentials below 0 mV. We recorded BK(Ca)-mediated currents from auditory inner hair cells (IHCs) in acutely isolated organs of Corti using the patch-clamp technique in whole-cell and excised patch configuration. In inside-out and outside-out patches, activation of BK(Ca) channels from IHCs showed the prototypic sensitivity to increased [Ca(2)(+)](i). However, channel activation at 0 [Ca(2)(+)](i) occurred at unusually negative potentials (half-maximal activation (V(h)) around 0 mV), indicating that a large fraction of the channels can be activated at physiological voltages without elevated [Ca(2)(+)](i). In intact IHCs, the activation curve of BK(Ca) currents recorded in whole-cell configuration exhibited a V(h) of -42 mV together with a high voltage dependence (slope factor of 10 mV) and submillisecond onset of current. Surprisingly, this activation was independent of changes in local [Ca(2)(+)](i) as shown by experiments that interfered with Ca(2)(+) influx through voltage-gated Ca(2)(+) (Cav) channels, release of Ca(2)(+) from internal stores, or intracellular buffer capacity. This behaviour is not due to beta-subunits of BK(Ca) (BKbeta), as genetic inactivation of the beta-subunit expressed in IHCs, KCNMB1, did not affect BK(Ca) gating. We conclude that the BK(Ca) channel protein in IHCs may be modified in order to rapidly activate and deactivate at resting [Ca(2)(+)](i). Our results suggest that BK(Ca) may function as a purely voltage-gated K(+) channel with exceptionally rapid activation kinetics, challenging the view that both increased cytosolic Ca(2)(+) and depolarization are generally required for activation of BK(Ca).

Animals↗

PubMed2005

Selective Golgi export of Kir2.1 controls the stoichiometry of functional Kir2.x channel heteromers.

Surface expression of ion channels and receptors often depends on intrinsic sequence motifs that control their intracellular transport along the secretory pathway. Although members of the Kir2.x subfamily share two such motifs - a diacidic ER export motif and a positively charged Golgi export motif - they strongly differ in their surface expression. Whereas Kir2.1 shows prominent plasma membrane localization, Kir2.4 channels accumulate within the Golgi complex. By constructing chimeras between Kir2.1 and Kir2.4 subunits, a stretch of 20 amino acids was identified in the Kir2.1 C-terminus that is both necessary and sufficient to promote anterograde transport of Kir channel subunits at the level of trafficking from the Golgi to the plasma membrane. The core element of the identified sequence bears a tyrosine-dependent YXXPhi consensus motif for adaptin binding, with the flanking residues determining its functional efficiency. As the signal is dominant in promoting surface transport of Kir2.1/Kir2.4 channel heteromers and is recognized by both the epithelial and neuronal intracellular sorting machinery, the preferential Golgi export of Kir2.1 will control the stoichiometry of Kir2.x heteromers expressed on the cell surface.

Amino Acid Motifs↗

PubMed2005

Cod106, a novel synaptic protein expressed in sensory hair cells of the inner ear and in CNS neurons.

The exquisite performance of the highly specialized mammalian inner ear requires a multitude of specific proteins. Yet, only a subset of these proteins has been identified and studied in detail. Here, we describe a novel gene expressed in the organ of Corti that encodes a membrane-associated protein of 106 kDa. The new protein, termed Cod106, lacks sequence homology to characterized gene products. As shown by in situ hybridization, it is expressed in auditory and vestibular hair cells, as well as in distinct sets of CNS neurons with particularly high abundance in hippocampus and cerebellum. Immunohistochemistry detected Cod106 at the basal, synaptic pole of cochlear outer hair cells and vestibular hair cells. In cultured hippocampal neurons, Cod106 immunofluorescence co-localized with the postsynaptic density protein 95 (PSD95), indicating a postsynaptic localization. Cell-type specificity and subcellular localization may be consistent with an involvement of Cod106 in synaptic processes.

Amino Acid Sequence↗

PubMed2004

Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating.

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.

Adenosine Triphosphate↗

PubMed2004

Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids.

Voltage-gated potassium (Kv) channels control action potential repolarization, interspike membrane potential, and action potential frequency in excitable cells. It is thought that the combinatorial association between distinct alpha and beta subunits determines whether Kv channels function as non-inactivating delayed rectifiers or as rapidly inactivating A-type channels. We show that membrane lipids can convert A-type channels into delayed rectifiers and vice versa. Phosphoinositides remove N-type inactivation from A-type channels by immobilizing the inactivation domains. Conversely, arachidonic acid and its amide anandamide endow delayed rectifiers with rapid voltage-dependent inactivation. The bidirectional control of Kv channel gating by lipids may provide a mechanism for the dynamic regulation of electrical signaling in the nervous system.

Animals↗

PubMed2004

Differential subunit composition of the G protein-activated inward-rectifier potassium channel during cardiac development.

Parasympathetic slowing of the heart rate is predominantly mediated by acetylcholine-dependent activation of the G protein-gated potassium (K+) channel (IK,ACh). This channel is composed of 2 inward-rectifier K+ (Kir) channel subunits, Kir3.1 and Kir3.4, that display distinct functional properties. Here we show that subunit composition of IK,ACh changes during embryonic development. At early stages, IK,ACh is primarily formed by Kir3.1, while in late embryonic and adult cells, Kir3.4 is the predominant subunit. This change in subunit composition results in reduced rectification of IK,ACh, allowing for marked K+ currents over the whole physiological voltage range. As a consequence, IK,ACh is able to generate the membrane hyperpolarization that underlies the strong negative chronotropy occurring in late- but not early-stage atrial cardiomyocytes upon application of muscarinic agonists. Both strong negative chronotropy and membrane hyperpolarization can be induced in early-stage cardiomyocytes by viral overexpression of the mildly rectifying Kir3.4 subunit. Thus, a switch in subunit composition is used to adopt IK,ACh to its functional role in adult cardiomyocytes.

Acetylcholine↗

PubMed2003

Resting potential and submembrane calcium concentration of inner hair cells in the isolated mouse cochlea are set by KCNQ-type potassium channels.

Cochlear inner hair cells (IHCs) transduce sound-induced vibrations into a receptor potential (RP) that controls afferent synaptic activity and, consequently, frequency and timing of action potentials in the postsynaptic auditory neurons. The RP is thought to be shaped by the two voltage-dependent K+ conductances, I(K,f) and I(K,s), that are carried by large-conductance Ca2+- and voltage-dependent K+ (BK)- and K(V)-type K+ channels. Using whole-cell voltage-clamp recordings in the acutely isolated mouse cochlea, we show that IHCs display an additional K+ current that is active at the resting membrane potential (-72 mV) and deactivates on hyperpolarization. It is potently blocked by the KCNQ-channel blockers linopirdine and XE991 but is insensitive to tetraethylammonium and 4-aminopyridine, which inhibit I(K,f) and I(K,s), respectively. Single-cell PCR and immunocytochemistry showed expression of the KCNQ4 subunit in IHCs. In current-clamp experiments, block of the KCNQ current shifted the resting membrane potential by approximately 7 to -65 mV and led to a significant activation of BK channels. Using BK channels as an indicator for submembrane intracellular Ca2+ concentration ([Ca2+]i), it is shown that the shift in IHC resting potential observed after block of the KCNQ channels leads to an increase in [Ca2+]i to values > or =1 microm. In conclusion, KCNQ channels set the resting membrane potential of IHCs in the isolated organ of Corti and thus maintain [Ca2+]i at low levels. Destabilization of the resting potential and increase in [Ca2+]i, as may result from impaired KCNQ4 function in IHCs, provide a novel explanation for the progressive hearing loss (DFNA2) observed in patients with defective KCNQ4 genes.

Animals↗

PubMed2003

Solution structure and function of the "tandem inactivation domain" of the neuronal A-type potassium channel Kv1.4.

Cumulative inactivation of voltage-gated (Kv) K(+) channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

Amino Acid Sequence↗

PubMed2002

Prestin, a new type of motor protein.

Prestin, a transmembrane protein found in the outer hair cells of the cochlea, represents a new type of molecular motor, which is likely to be of great interest to molecular cell biologists. In contrast to enzymatic-activity-based motors, prestin is a direct voltage-to-force converter, which uses cytoplasmic anions as extrinsic voltage sensors and can operate at microsecond rates. As prestin mediates changes in outer hair cell length in response to membrane potential variations, it might be responsible for sound amplification in the mammalian hearing organ.

Animals↗

PubMed2001

A helical region in the C terminus of small-conductance Ca2+-activated K+ channels controls assembly with apo-calmodulin.

Small conductance Ca(2+)-activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca(2+) in the submicromolar range. This high affinity for Ca(2+) results from Ca(2+)-independent association of the SK alpha-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396-487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca(2+)-free CaM, and results in SK channels that are no longer gated by Ca(2+). The results show that the Ca(2+)-independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaM-interacting proteins.

Amino Acid Sequence↗

PubMed1998

Fast Inactivation of Voltage-Gated K(+) Channels: From Cartoon to Structure.

Fast inactivation of voltage-gated potassium (K(v)) channels is the best understood gating transition in ion channels and is brought about by an NH(2)-terminal domain (ball domain) of the channel's alpha-subunit, which physically blocks the open pore. Recent analysis by nuclear magnetic resonance spectroscopy showed that ball domains from various K(v) channels exhibit well-defined but distinct structures in aqueous solution.

Journal Article↗

PubMedDate not supplied

Prestin, the motor protein of outer hair cells.

Prestin is a gene recently cloned from mammalian cochlear outer hair cells (OHC) using a single cell type, outer minus inner hair cell, specific suppressive subtractive hybridization procedure. The localization and gene expression profile of the prestin protein fits the pattern of OHC's development of electromotility. When prestin is abundantly expressed in normally nonmotile kidney cells, nonlinear capacitance and motility that are normally only seen in OHCs can be recorded. Furthermore, both nonlinear capacitance and motility can be reduced by salicylate, a well-known inhibitor of electromotility. These data suggest that prestin is the motor protein of OHCs. Amino acid sequence and gene structure analysis indicate that prestin is the fifth member of a newly discovered anion transport family (SLC26) that includes PDS, DRA and DTDST, which are chloride-iodide transporters, Cl-/HCO3- exchangers or sulfate transporters. Prestin shares overall structure similarity with this anion transporter family. Recently, intracellular anions (chloride or bicarbonate) were found to be essential for OHC electromotility and prestin's function.

Animals↗