KCNQ4 channels expressed in mammalian cells: functional characteristics and pharmacology

Human cloned KCNQ4 channels were stably expressed in HEK-293 cells and characterized with respect to function and pharmacology. Patch-clamp measurements showed that the KCNQ4 channels conducted slowly activating currents at potentials more positive than -60 mV. From the Boltzmann function fitted to the activation curve, a half-activation potential of -32 mV and an equivalent gating charge of 1.4 elementary charges was determined. The instantaneous current-voltage relationship revealed strong inward rectification. The KCNQ4 channels were blocked in a voltage-independent manner by the memory-enhancing M current blockers XE-991 and linopirdine with IC(50) values of 5.5 and 14 microM, respectively. The antiarrhythmic KCNQ1 channel blocker bepridil inhibited KCNQ4 with an IC(50) value of 9.4 microM, whereas clofilium was without significant effect at 100 microM. The KCNQ4-expressing cells exhibited average resting membrane potentials of -56 mV in contrast to -12 mV recorded in the nontransfected cells. In conclusion, the activation and pharmacology of KCNQ4 channels resemble those of M currents, and it is likely that the function of the KCNQ4 channel is to regulate the subthreshold electrical activity of excitable cells.     

Evolutionary convergence of alternative splicing in ion channels

In Drosophila melanogaster and humans, members of three different ion-channel gene families share tandem exon duplications, which are alternatively spliced. In this article, I demonstrate that the duplication events that give rise to these mutually exclusive exons are unlikely to be ancestral but have probably occurred independently in different lineages. These events provide remarkable examples of evolutionary convergence in alternative splicing. The result has important implications for the analysis of regulation of alternative splicing using comparative genomics and our understanding of molecular evolution.     

Increase of functional diversity by alternative splicing

A large-scale analysis of protein isoforms arising from alternative splicing shows that alternative splicing tends to insert or delete complete protein domains more frequently than expected by chance, whereas disruption of domains and other structural modules is less frequent. If domain regions are disrupted, the functional effect, as predicted from 3D structure, is frequently equivalent to removal of the entire domain. Also, short alternative splicing events within domains, which might preserve folded structure, target functional residues more frequently than expected. Thus, it seems that positive selection has had a major role in the evolution of alternative splicing.     

Ionic permeation and conduction properties of neuronal KCNQ2/KCNQ3 potassium channels

Heteromeric KCNQ2/3 potassium channels are thought to underlie the M-current, a subthreshold potassium current involved in the regulation of neuronal excitability. KCNQ channel subunits are structurally unique, but it is unknown whether these structural differences result in unique conduction properties. Heterologously expressed KCNQ2/3 channels showed a permeation sequence of while showing a conduction sequence of A differential contribution of component subunits to the properties of heteromeric KCNQ2/3 channels was demonstrated by studying homomeric KCNQ2 and KCNQ3 channels, which displayed contrasting ionic selectivities. KCNQ2/3 channels did not exhibit an anomalous mole-fraction effect in mixtures of K(+) and Rb(+). However, extreme voltage-dependence of block by external Cs(+) was indicative of multi-ion pore behavior. Block of KCNQ2/3 channels by external Ba(2+) ions was voltage-independent, demonstrating unusual ionic occupation of the outer pore. Selectivity properties and block of KCNQ2 were altered by mutation of outer pore residues in a manner consistent with the presence of multiple ion-binding sites. KCNQ2/3 channel deactivation kinetics were slowed exclusively by Rb(+), whereas activation of KCNQ2/3 channels was altered by a variety of external permeant ions. These data indicate that KCNQ2/3 channels are multi-ion pores which exhibit distinctive mechanisms of ion conduction and gating.     

Redox-dependent structural ambivalence of the cytoplasmic domain in the inner ear-specific cadherin 23 isoform

Cadherin 23 (Cdh23), an essential factor in inner ear mechano-electric transduction, exists in two alternatively spliced forms, Cdh23(+68) and Cdh23(−68), depending on the presence and absence of exon 68. Cdh23(+68) is inner ear-specific. The exon 68-corresponding region confers an α-helical configuration upon the cytoplasmic domain (Cy) and includes a cysteine residue, Cys³²⁴⁰. We demonstrate here that Cy(+68) as well as the transmembrane (TM) plus Cy(+68) region is present in two different forms in transfected cells, reduced and non-reduced, the latter existing in more compact configuration than the former. The observed characteristic of Cy(+68) was completely abolished by Cys³²⁴⁰Ala substitution. Treatment of TMCy(+68)-transfected cells with diethyl maleate, a glutathione depleting reagent, resulted in conversion of the non-reduced to the reduced form of TMCy(+68), suggesting glutathione to be a Cys³²⁴⁰-binding partner. Multiple alignment of mammalian Cdh23Cy sequences indicated the occurrence of conformation-inducible Cys in Cdh23Cy of mammals, but not lower vertebrates. The implications of Cys-dependent structural ambivalence of Cdh23 in inner ear mechanosensation are discussed.     

TMEM16A alternative splicing isoforms in Xenopus tropicalis: Distribution and functional properties

Oocytes of Xenopus tropicalis elicit a Ca²⁺-dependent outwardly rectifying, low-activating current (I_Cl,Ca) that is inhibited by Cl⁻ channel blockers. When inactivated, I_Cl,Ca shows an exponentially decaying tail current that is related to currents generated by TMEM16A ion channels. Accordingly, RT-PCR revealed the expression of five alternatively spliced isoforms of TMEM16A in oocytes, which, after expression in HEK-293 cells, gave rise to fully functional Cl⁻ channels. Upon hyperpolarization to −80 mV a transient current was observed only in isoforms that carry the exon 1d, coding for two potentially phosphorylatable Threonine residues. The identified isoforms are differentially expressed in several tissues of the frog. Thus, it appears that X. tropicalis oocytes express TMEM16A that gives rise to a Ca²⁺-dependent Cl⁻ current, which is different from the previously reported voltage-dependent outwardly rectifying Cl⁻ current.     

Structural and functional diversity generated by alternative mRNA splicing

Alternative mRNA splicing is becoming increasingly recognized as an important mechanism for the generation of structural and functional diversity in proteins. Recent estimations predict that approximately 50% of all eukaryotic proteins can be alternatively spliced. Several lines of evidence suggest that alternative mRNA splicing results in small changes in protein structure and is likely to fine-tune the function and specificity of the affected protein. However, knowledge of how alternative splicing regulates cellular processes on the molecular level is still limited. It is only recently that structures of alternatively spliced proteins have been solved. These studies have shown that alternative splicing affects the structure not only in the vicinity of the splice site but also at long distance.     

Surface expression and single channel properties of KCNQ2/KCNQ3, M-type K+ channels involved in epilepsy

Mutations in either KCNQ2 or KCNQ3 underlie benign familial neonatal convulsions (BFNC), an inherited epilepsy. The corresponding proteins are co-expressed in broad regions of the brain and associate to heteromeric K(+) channels. These channels mediate M-type currents that regulate neuronal excitability. We investigated the basis for the increase in currents seen after co-expressing these subunits in Xenopus oocytes. Noise analysis and single channel recordings revealed a conductance of approximately 18 pS for KCNQ2 and approximately 7 pS for KCNQ3. Different conductance levels (ranging from 8 to 22 pS) were seen upon co-expression. Their weighted average is close to that obtained by noise analysis (16 pS). The open probability of heteromeric channels was not increased significantly. Co-expression of both subunits increased the surface expression of KCNQ2 and KCNQ3 by factors of 5 and >10, respectively. A KCNQ2 mutant associated with BFNC that has a truncated cytoplasmic carboxyl terminus did not reach the surface and failed to stimulate KCNQ3 surface expression. By contrast, several BFNC-associated missense mutations in KCNQ2 or KCNQ3 did not alter their surface expression. Thus, the increase in currents seen upon co-expressing KCNQ2 and KCNQ3 is predominantly due to an increase in surface expression, which is dependent on an intact carboxyl terminus.     

Genome-wide association between branch point properties and alternative splicing

The branch point (BP) is one of the three obligatory signals required for pre-mRNA splicing. In mammals, the degeneracy of the motif combined with the lack of a large set of experimentally verified BPs complicates the task of modeling it in silico, and therefore of predicting the location of natural BPs. Consequently, BPs have been disregarded in a considerable fraction of the genome-wide studies on the regulation of splicing in mammals. We present a new computational approach for mammalian BP prediction. Using sequence conservation and positional bias we obtained a set of motifs with good agreement with U2 snRNA binding stability. Using a Support Vector Machine algorithm, we created a model complemented with polypyrimidine tract features, which considerably improves the prediction accuracy over previously published methods. Applying our algorithm to human introns, we show that BP position is highly dependent on the presence of AG dinucleotides in the 3' end of introns, with distance to the 3' splice site and BP strength strongly correlating with alternative splicing. Furthermore, experimental BP mapping for five exons preceded by long AG-dinucleotide exclusion zones revealed that, for a given intron, more than one BP can be chosen throughout the course of splicing. Finally, the comparison between exons of different evolutionary ages and pseudo exons suggests a key role of the BP in the pathway of exon creation in human. Our computational and experimental analyses suggest that BP recognition is more flexible than previously assumed, and it appears highly dependent on the presence of downstream polypyrimidine tracts. The reported association between BP features and the splicing outcome suggests that this, so far disregarded but yet crucial, element buries information that can complement current acceptor site models.     

Determinants within the turret and pore-loop domains of KCNQ3 K+ channels governing functional activity

KCNQ1-5 (Kv7.1-7.5) subunits assemble to form a variety of functional K⁺ channels in the nervous system, heart, and epithelia. KCNQ1 and KCNQ4 homomers and KCNQ2/3 heteromers yield large currents, whereas KCNQ2 and KCNQ3 homomers yield small currents. Since the unitary conductance of KCNQ3 is five- to 10-fold greater than that of KCNQ4 or KCNQ1, these differences are even more striking. To test for differential membrane protein expression, we performed biotinylation and total internal reflection fluorescence imaging assays; however, both revealed only small differences among the channels, leading us to investigate other mechanisms at work. We probed the molecular determinants governing macroscopic current amplitudes, with focus on the turret and pore-loop domains of KCNQ1 and KCNQ3. Elimination of the putative N289 glycosylation site in KCNQ1 reduced current density by ∼56%. A chimera consisting of KCNQ3 with the turret domain (TD) of KCNQ1 increased current density by about threefold. Replacement of the proximal half of the TD in KCNQ3 with that of KCNQ1 increased current density by fivefold. A triple chimera containing the TD of KCNQ1 and the carboxy terminus of KCNQ4 yielded current density 10- or sixfold larger than wild-type KCNQ3 or KCNQ1, respectively, suggesting that the effects on current amplitudes of the TD and the carboxy-terminus are additive. Critical was the role of the intracellular TEA⁺-binding site. The KCNQ3 (A315T) swap increased current density by 10-fold, and the converse KCNQ1 (T311A) swap reduced it by 10-fold. KCNQ3 (A315S) also yielded greatly increased current amplitudes, whereas currents from mutant A315V channels were very small. The KCNQ3 (A315T) mutation increased the sensitivity of the channels to external Ba²⁺ block by eight- to 28-fold, consistent with this mutation altering the structure of the selectivity filter. To investigate a structural hypothesis for the effects of these mutations, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels, using KvAP as a template. The modeling suggests a critical stabilizing interaction between the pore helix and the selectivity filter that is absent in wild-type KCNQ3 and the A315V mutant, but present in the A315T and A315S mutants. We conclude that KCNQ3 homomers are well expressed at the plasma membrane, but that most wild-type channels are functionally silent, with rearrangements of the pore-loop architecture induced by the presence of a hydroxyl-containing residue at the 315 position “unlocking” the channels into a conductive conformation.     

Exon-specific RNAi: a tool for dissecting the functional relevance of alternative splicing

The goal of functional genomics is to determine the function of each protein encoded by an organism. Typically, this is done by inactivating individual genes and, subsequently, analyzing the phenotype of the modified organisms. In higher eukaryotes, where a tremendous amount of alternative splicing occurs, such approaches are not feasible because they have the potential to simultaneously affect multiple proteins that could have quite distinct and important functions. Thus, it is necessary to develop techniques that inactivate only a subset of proteins synthesized from genes encoding alternatively spliced mRNAs. Here we demonstrate that RNA interference (RNAi) can be used to selectively degrade specific alternatively spliced mRNA isoforms in cultured Drosophila cells. This is achieved by treating the cells with double-stranded RNA corresponding to an alternatively spliced exon. This technique may prove to be a powerful tool to assess the function of proteins synthesized from alternatively spliced mRNAs. In addition, these results have implications regarding the mechanism of RNAi in Drosophila.