Robert Bähring

Conserved Kv4 N-terminal Domain Critical for Effects of Kv Channel-interacting Protein 2.2 on Channel Expression and Gating

Association of Kv channel-interacting proteins (KChIPs) with Kv4 channels leads to modulation of these A-type potassium channels (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553–556). We cloned a KChIP2 splice variant (KChIP2.2) from human ventricle. In comparison with KChIP2.1, coexpression of KChIP2.2 with human Kv4 channels in mammalian cells slowed the onset of Kv4 current inactivation (2–3-fold), accelerated the recovery from inactivation (5–7-fold), and shifted Kv4 steady-state inactivation curves by 8–29 mV to more positive potentials. The features of Kv4.2/KChIP2.2 currents closely resemble those of cardiac rapidly inactivating transient outward currents. KChIP2.2 stimulated the Kv4 current density in Chinese hamster ovary cells by ∼55-fold. This correlated with a redistribution of immunoreactivity from perinuclear areas to the plasma membrane. Increased Kv4 cell-surface expression and current density were also obtained in the absence of KChIP2.2 when the highly conserved proximal Kv4 N terminus was deleted. The same domain is required for association of KChIP2.2 with Kv4 α-subunits. We propose that an efficient transport of Kv4 channels to the cell surface depends on KChIP binding to the Kv4 N-terminal domain. Our data suggest that the binding is necessary, but not sufficient, for the functional activity of KChIPs.

Functional and molecular aspects of voltage-gated K+ channel beta subunits

ABSTRACT: Voltage‐gated potassium channels (Kv) of the Shaker‐related superfamily are assembled from membrane‐integrated α subunits and auxiliary β subunits. The β subunits may increase Kv channel surface expression and/or confer A‐type behavior to noninactivating Kv channels in heterologous expression systems. The interaction of Kvα and Kvβ subunits depends on the presence or absence of several domains including the amino‐terminal N‐type inactivating and NIP domains and the Kvα and Kvβ binding domains. Loss of function of Kvβ1.1 subunits leads to a reduction of A‐type Kv channel activity in hippocampal and striatal neurons of knock‐out mice. This reduction may be correlated with altered cognition and motor control in the knock‐out mice.

Kinetic analysis of open‐ and closed‐state inactivation transitions in human Kv4.2 A‐type potassium channels

1 We studied the gating kinetics of Kv4.2 channels, the molecular substrate of neuronal somatodendritic A‐type currents. For this purpose wild‐type and mutant channels were transiently expressed in the human embryonic kidney (HEK) 293 cell line and currents were measured in the whole‐cell patch‐clamp configuration. 2 Kv4.2 channels inactivated from pre‐open closed state(s) with a mean time constant of 959 ms at ‐50 mV. This closed‐state inactivation was not affected by a deletion of the Kv4.2 N‐terminus (Δ2‐40). 3 Kv4.2 currents at +40 mV inactivated with triple‐exponential kinetics. A fast component (τ= 11 ms) accounted for 73 %, an intermediate component (τ= 50 ms) for 23 % and a slow component (τ= 668 ms) for 4 % of the total decay. 4 Both the fast and the intermediate components of inactivation were slowed by a deletion of the Kv4.2 N‐terminus (τ= 35 and 111 ms) and accounted for 33 and 56 %, respectively, of the total decay. The slow component was moderately accelerated by the truncation (τ= 346 ms) and accounted for 11 % of the total Kv4.2 current inactivation. 5 Recovery from open‐state inactivation and recovery from closed‐state inactivation occurred with similar kinetics in a strongly voltage‐dependent manner. Neither recovery reaction was affected by the N‐terminal truncation. 6 Kv4.2 Δ2‐40 channels displayed slowed deactivation kinetics, suggesting that the N‐terminal truncation leads to a stabilization of the open state. 7 Simulations with an allosteric model of inactivation, supported by the experimental data, suggested that, in response to membrane depolarization, Kv4.2 channels accumulate in the closed‐inactivated state(s), from which they directly recover, bypassing the open state.

Activated radixin is essential for GABAA receptor α5 subunit anchoring at the actin cytoskeleton

Neurotransmitter receptor clustering is thought to represent a critical parameter for neuronal transmission. Little is known about the mechanisms that anchor and concentrate inhibitory neurotransmitter receptors in neurons. GABAA receptor (GABAAR) α5 subunits mainly locate at extrasynaptic sites and are thought to mediate tonic inhibition. Notably, similar as synaptic GABAARs, these receptor subtypes also appear in cluster formations at neuronal surface membranes and are of particular interest in cognitive processing. GABAAR α5 mutation or depletion facilitates trace fear conditioning or improves spatial learning in mice, respectively. Here, we identified the actin‐binding protein radixin, a member of the ERM family, as the first directly interacting molecule that anchors GABAARs at cytoskeletal elements. Intramolecular activation of radixin is a functional prerequisite for GABAAR α5 subunit binding and both depletion of radixin expression as well as replacement of the radixin F‐actin binding motif interferes with GABAAR α5 cluster formation. Our data suggest radixin to represent a critical factor in receptor localization and/or downstream signaling.

Permeation and block of rat glur6 glutamate receptor channels by internal and external polyamines

1 Polyamine block of rat GluR6(Q) glutamate receptor channels was studied in outside‐out patches from transiently transfected HEK 293 cells. With symmetrical 150 mm Na+ and 30 μm internal spermine there was biphasic voltage dependence with 95% block at +40 mV but only 20% block at +140mV. Dose–inhibition analysis for external spermine also revealed biphasic block; the Ka at +40 mV (54 μm) was lower than at +80 (167μm) and –80 mV (78 μm). 2 For internal polyamines relief from block was most pronounced for spermine, weaker for N‐(4‐hydroxyphenylpropanoyl)‐spermine (PPS), and virtually absent for philanthotoxin 343 (PhTX 343), suggesting that permeation of polyamines varies with cross‐sectional width (spermine, 0.44 nm; PPS, 0.70 nm; PhTX 343, 0.75 nm). 3 With putrescine, spermidine, or spermine as sole external cations, inward currents at –120 mV confirmed permeation of polyamines. For bi‐ionic conditions with 90 mm polyamine and 150 mm Na+i reversal potentials were –12.4 mV for putrescine (permeability ratio relative to Na+, PPut/PNa= 0.42) and –32.7 mV for spermidine (PSpd/PNa= 0.07). Currents carried by spermine were too small to analyse accurately in the majority of patches. 4 Increasing [Na+]i from 44 to 330 mm had no effect on the potential for 50% block (V½) by 30 μm internal spermine; however, relief from block at positive membrane potentials increased with [Na+]i. In contrast, raising [Na+]o from 44 to 330 mm resulted in a depolarizing shift in V½, indicating a strong interaction between internal polyamines and external per meant ions. 5 The Woodhull infinite barrier model of ion channel block adequately described the action of spermine at membrane potentials insufficient to produce relief from block. For 30 μm internal spermine such analysis gave Kd(0)= 2.5 μm, z θ= 1.97; block by 30 μm external spermine was weaker and less voltage dependent (Kd(0)= 37.8 μm and zδ= 0.55); δ and θ are electrical distances measured from the outside and inside, respectively. 6 Fits of the Woodhull equation for a permeable blocker adequately described both onset and relief from block by spermine over a wide range of membrane potentials. However, the rate constants and zδ values estimated for block by internal spermine predicted much stronger external block than was measured experimentally, and vice versa. 7 An Eyring rate theory model with two energy wells and three barriers explained qualitatively many characteristic features of the action of polyamines on GluPvs, including biphasic I–V relationships, weaker block by external than internal spermine and low permeability.

N-type Inactivation Features of Kv4.2 Channel Gating

We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Delta40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K+. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Delta40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Delta40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.

Mechanisms of closed-state inactivation in voltage-gated ion channels

Abstract  Inactivation of voltage‐gated ion channels is an intrinsic auto‐regulatory process necessary to govern the occurrence and shape of action potentials and establish firing patterns in excitable tissues. Inactivation may occur from the open state (open‐state inactivation, OSI) at strongly depolarized membrane potentials, or from pre‐open closed states (closed‐state inactivation, CSI) at hyperpolarized and modestly depolarized membrane potentials. Voltage‐gated Na+, K+, Ca2+ and non‐selective cationic channels utilize both OSI and CSI. Whereas there are detailed mechanistic descriptions of OSI, much less is known about the molecular basis of CSI. Here, we review evidence for CSI in voltage‐gated cationic channels (VGCCs) and recent findings that shed light on the molecular mechanisms of CSI in voltage‐gated K+ (Kv) channels. Particularly, complementary observations suggest that the S4 voltage sensor, the S4S5 linker and the main S6 activation gate are instrumental in the installment of CSI in Kv4 channels. According to this hypothesis, the voltage sensor may adopt a distinct conformation to drive CSI and, depending on the stability of the interactions between the voltage sensor and the pore domain, a closed‐inactivated state results from rearrangements in the selectivity filter or failure of the activation gate to open. Kv4 channel CSI may efficiently exploit the dynamics of the subthreshold membrane potential to regulate spiking properties in excitable tissues.

Coexpression of the KCNA3B Gene Product with Kv1.5 Leads to a Novel A-type Potassium Channel

Shaker-related voltage-gated potassium (Kv) channels may be heterooligomers consisting of membrane-integral α-subunits associated with auxiliary cytoplasmic β-subunits. In this study we have cloned the human Kvβ3.1 subunit and the corresponding KCNA3B gene. Identification of sequence-tagged sites in the gene mapped KCNA3B to band p13.1 of human chromosome 17. Comparison of the KCNA1B,KCNA2B, and KCNA3B gene structures showed that the three Kvβ genes have very disparate lengths varying from ≥350 kb (KCNA1B) to ∼7 kb (KCNA3B). Yet, the exon patterns of the three genes, which code for the seven known mammalian Kvβ subunits, are very similar. The Kvβ1 and Kvβ2 splice variants are generated by alternative use of 5′-exons. Mouse Kvβ4, a potential splice variant of Kvβ3, is a read-through product where the open reading frame starts within the sequence intervening between Kvβ3 exons 7 and 8. The human KCNA3B sequence does not contain a mouse Kvβ4-like open reading frame. Human Kvβ3 mRNA is specifically expressed in the brain, where it is predominantly detected in the cerebellum. The heterologous coexpression of human Kv1.5 and Kvβ3.1 subunits in Chinese hamster ovary cells yielded a novel Kv channel mediating very fast inactivating (A-type) outward currents upon depolarization. Thus, the expression of Kvβ3.1 subunits potentially extends the possibilities to express diverse A-type Kv channels in the human brain.

An analysis of philanthotoxin block for recombinant rat GluR6(Q) glutamate receptor channels

1 The action of philanthotoxin 343 (PhTX) on rat homomeric GluR6(Q) recombinant glutamate receptor channels was analysed using concentration‐jump techniques and outside‐out patches from HEK 293 cells. Both onset and recovery from block by external PhTX were dependent on the presence of agonist, indicating that channels must open for PhTX to bind and that channel closure can trap PhTX. 2 Block by external PhTX developed with double‐exponential kinetics. The rate of onset of the fast component of block showed an exponential increase per 27 mV hyperpolarization over the range ‐40 to ‐100 mV. The rate of onset of the slow component of block showed a non‐linear concentration dependence indicating a rate‐limiting step in the blocking mechanism. 3 The extent of block by 1 μM external PhTX was maximal at ‐40 mV and did not increase with further hyperpolarization; the rate of recovery from block by external PhTX increased 6‐fold on hyperpolarization from ‐40 to ‐100 mV suggesting that PhTX permeates at negative membrane potentials. 4 Apparent Kd values for block by external PhTX estimated from dose‐inhibition experiments decreased 300‐fold on hyperpolarization from +40 mV (Kd, 19.6 μM) to ‐40 mV (Kd, 69 nM); there was little further increase in affinity with hyperpolarization to ‐80 mV (Kd, 56 nM), consistent with permeation of PhTX at negative membrane potentials. 5 Block by internal PhTX showed complex kinetics and voltage dependence. Analysis with voltage ramps from ‐120 to +120 mV indicated a Kd at 0 mV of 20 μM, decreasing e‐fold per 16 mV depolarization. However, at +90 mV the extent of block by 1 and 10 μM internal PhTX (73 % and 95 %, respectively) reached a maximum and did not increase with further depolarization. 6 Voltage‐jump analysis of block by 100 μM internal PhTX revealed partial trapping. With 100 ms jumps from ‐100 to ‐40 mV, onset and recovery from block were complete within 5 ms. With jumps of longer duration the extent of block increased, with a time constant of 8.1 s, reaching 84 % at 30 s. On repolarization to ‐100 mV, recovery from block showed fast and slow components. 7 The amplitude of the slow component of block by internal PhTX showed a biphasic voltage dependence, first increasing then decreasing with progressive depolarization. Maximum block was obtained at 0 mV. 8 Our results suggest that PhTX acts as an open channel blocker; however, provided that the toxin remains bound to the channel, an allosteric mechanism destabilizes the open state, inducing channel closing and trapping PhTX. Strong depolarization for internal PhTX, or strong hyperpolarization for external PhTX, forces the toxin to permeate before it triggers entry into closed blocked states.

Contribution of N- and C-terminal channel domains to Kv channel interacting proteins in a mammalian cell line

Association of Shal gene‐related voltage‐gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C‐terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine‐scanning mutagenesis within the proximal 40 amino acid portion and a structure‐based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C‐terminus was truncated at various positions. Wild‐type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2‐induced modulation of Kv4.2 currents was studied with whole‐cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N‐terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C‐terminal deletions also had marked effects on KChIP2‐dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C‐terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose ‘anchoring’ at T1‐ and C‐terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.