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Wild-type Channels (wild-type + channel)

Distribution by Scientific Domains

Life Sciences	80%

Selected Abstracts

Episodic ataxia type 1 mutations in the KCNA1 gene impair the fast inactivation properties of the human potassium channels Kv1.4-1.1/Kv,1.1 and Kv1.4-1.1/Kv,1.2

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006
Paola Imbrici
Abstract Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by constant muscle rippling movements (myokymia) and episodic attacks of ataxia. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene KCNA1 (hKv1.1), which alter the delayed-rectifier function of the channel. Shaker -like channels of different cell types may be formed by unique hetero-oligomeric complexes comprising Kv1.1, Kv1.4 and Kv,1.x subunits. Here we show that the human Kv,1.1 and Kv,1.2 subunits modulated the functional properties of tandemly linked Kv1.4-1.1 wild-type channels expressed in Xenopus laevis oocytes by (i) increasing the rate and amount of N-type inactivation, (ii) slowing the recovery rate from inactivation, (iii) accelerating the cumulative inactivation of the channel and (iv) negatively shifting the voltage dependence of inactivation. To date, the role of the human Kv1.4-1.1, Kv1.4-1.1/Kv,1.1 and Kv1.4-1.1/Kv,1.2 channels in the aetiopathogenesis of EA1 has not been investigated. Here we also show that the EA1 mutations E325D, V404I and V408A, which line the ion-conducting pore, and I177N, which resides within the S1 segment, alter the fast inactivation and repriming properties of the channels by decreasing both the rate and degree of N-type inactivation and by accelerating the recovery from fast inactivation. Furthermore, the E325D, V404I and I177N mutations shifted the voltage dependence of the steady-state inactivation to more positive potentials. The results demonstrate that the human Kv,1.1 and Kv,1.2 subunits regulate the proportion of wild-type Kv1.4-1.1 channels that are available to open. Furthermore, EA1 mutations alter heteromeric channel availability which probably modifies the integration properties and firing patterns of neurones controlling cognitive processes and body movements. [source]

The Drosophila cacts2 mutation reduces presynaptic Ca2+ entry and defines an important element in Cav2.1 channel inactivation

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2006
G. T. Macleod
Abstract Voltage-gated Ca2+ channels in nerve terminals open in response to action potentials and admit Ca2+, the trigger for neurotransmitter release. The cacophony gene encodes the primary presynaptic voltage-gated Ca2+ channel in Drosophila motor-nerve terminals. The cacts2 mutant allele of cacophony is associated with paralysis and reduced neurotransmission at non-permissive temperatures but the basis for the neurotransmission deficit has not been established. The cacts2 mutation occurs in the cytoplasmic carboxyl tail of the ,1 -subunit, not within the pore-forming trans-membrane domains, making it difficult to predict the mutation's impact. We applied a Ca2+ -imaging technique at motor-nerve terminals of mutant larvae to test the hypothesis that the neurotransmission deficit is a result of impaired Ca2+ entry. Presynaptic Ca2+ signals evoked by single and multiple action potentials showed a temperature-dependent reduction. The amplitude of the reduction was sufficient to account for the neurotransmission deficit, indicating that the site of the cacts2 mutation plays a role in Ca2+ channel activity. As the mutation occurs in a motif conserved in mammalian high-voltage-activated Ca2+ channels, we used a heterologous expression system to probe the effect of this mutation on channel function. The mutation was introduced into rat Cav2.1 channels expressed in human embryonic kidney cells. Patch-clamp analysis of mutant channels at the physiological temperature of 37 °C showed much faster inactivation rates than for wild-type channels, demonstrating that the integrity of this motif is critical for normal Cav2.1 channel inactivation. [source]

Dominance of the lurcher mutation in heteromeric kainate and AMPA receptor channels

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2001
Martin K. Schwarz
Abstract Homomeric glutamate receptor (GluR) channels become spontaneously active when the last alanine residue within the invariant SYTANLAAF-motif in the third membrane segment is substituted by threonine. The same mutation in the orphan GluR,2 channel is responsible for neurodegeneration in ,Lurcher' (Lc) mice. Since most native GluRs are composed of different subunits, we investigated the effect of an Lc-mutated subunit in heteromeric kainate and AMPA receptors expressed in HEK293 cells. Kainate receptor KA2 subunits, either wild type or carrying the Lc mutation (KA2Lc), are retained inside the cell but are surface-expressed when assembled with GluR6 sununits. Importantly, KA2Lc dominates the gating of KA2Lc/GluR6WT channels, as revealed by spontaneous activation and by slowed desensitization and deactivation kinetics of ligand-activated whole-cell currents. Moreover, the AMPA receptor subunit GluR-BLc(Q) which forms spontaneously active homomeric channels with rectifying current-voltage relationships, dominates the gating of heteromeric GluR-BLc(Q)/GluR-A(R) channels. The spontaneous currents of these heteromeric AMPAR channels show linear current,voltage relationships, and the ligand-activated whole-cell currents display slower deactivation and desensitization kinetics than the respective wild-type channels. For heteromeric Lc-mutated kainate and AMPA receptors, the effects on kinetics were reduced relative to the homomeric Lc-mutated forms. Thus, an Lc-mutated subunit can potentially influence heteromeric channel function in vivo, and the severity of the phenotype will critically depend on the levels of homomeric GluRLc and heteromeric GluRLc/GluRWT channels. [source]

Mechanisms by which atrial fibrillation-associated mutations in the S1 domain of KCNQ1 slow deactivation of IKs channels

THE JOURNAL OF PHYSIOLOGY, Issue 17 2008
Lioara Restier
The slow delayed rectifier K+ current (IKs) is a major determinant of action potential repolarization in the heart. IKs channels are formed by coassembly of pore-forming KCNQ1 ,-subunits and ancillary KCNE1 ,-subunits. Two gain of function mutations in KCNQ1 subunits (S140G and V141M) have been associated with atrial fibrillation (AF). Previous heterologous expression studies found that both mutations caused IKs to be instantaneously activated, presumably by preventing channel closure. The purpose of this study was to refine our understanding of the channel gating defects caused by these two mutations located in the S1 domain of KCNQ1. Site-directed mutagenesis was used to replace S140 or V141 with several other natural amino acids. Wild-type and mutant channels were heterologously expressed in Xenopus oocytes and channel function was assessed with the two-microelectrode voltage clamp technique. Long intervals between voltage clamp pulses revealed that S140G and V141M KCNQ1-KCNE1 channels are not constitutively active as previously reported, but instead exhibit extremely slow deactivation. The slow component of IKs deactivation was decreased 62-fold by S140G and 140-fold by the V141M mutation. In addition, the half-point for activation of these mutant IKs channels was ,50 mV more negative than wild-type channels. Other substitutions of S140 or V141 in KCNQ1 caused variable shifts in the voltage dependence of activation, but slowed IKs deactivation to a much lesser extent than the AF-associated mutations. Based on a published structural model of KCNQ1, S140 and V141 are located near E160 in S2 and R237 in S4, two charged residues that could form a salt bridge when the channel is in the open state. In support of this model, mutational exchange of E160 and R237 residues produced a constitutively open channel. Together our findings suggest that altered charge-pair interactions within the voltage sensor module of KCNQ1 subunits may account for slowed IKs deactivation induced by S140 or V141. [source]

Effect of K+ and Rb+ on the action of verapamil on a voltage-gated K+ channel, hKv1.3: implications for a second open state?

BRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2009
Z Kuras
Background and purpose:, Verapamil blocks current through the voltage-gated K+ channel Kv1.3 in the open and inactivated state of the channel but not the closed state. The binding site for verapamil was proposed to be close to the selectivity filter and the occupancy of the selectivity filter might therefore influence verapamil affinity. Experimental approach:, We investigated the influence of intra- and extracellular K+ and Rb+ on the effect of verapamil by patch-clamp studies, in COS-7 cells transfected with hKv1.3 channels. Key results:, Verapamil affinity was highest in high intracellular K+ concentrations ([K+]i) and lowest in low [Rb+]i, indicating an influence of intracellular cations on verapamil affinity. Experiments with a mutant channel (H399T), exhibiting a strongly reduced C-type inactivated state, demonstrated that part of this changed verapamil affinity in wild-type channels could be caused by altered C-type inactivation. External K+ and Rb+ could influence verapamil affinity by a voltage-dependent entry into the channel thereby modifying the verapamil off-rate and in addition causing a voltage-dependent verapamil off-rate. Conclusions and implications:, Recovery from verapamil block was mainly due to the voltage-dependent closing of channels (state-dependent block), implying a second open state of the channel. This hypothesis was confirmed by the dependency of the tail current time course on duration of the prepulse. We conclude that the wild-type hKv1.3 channel undergoes at least two different conformational changes before finally closing with a low verapamil affinity in one open state and a high verapamil affinity in the other open state. [source]