Channel Pore Region Research Articles

Ion Permeation Efficiency through Potassium Channels

Potassium channels underlie important physiological functions such as cellular ionic homeostasis and nerve signal transduction. Alongside the discrimination of potassium over sodium, the channels' capability of finely tuning their permeability for potassium is crucial for their function. In most potassium channels, these two features are combined in the selectivity filter, a narrow region at the outer mouth of the channel that is permeated by potassium ions in a single file. Here we present our findings from simulations of the wild-type and mutants of the bacterial KcsA potassium channel pore-region under near-physiological trans-membrane voltages. The simulations accurately reproduce important electrophysiological parameters of these KcsA variants, such as peak conductance and rectification. Based on the recording of thousands of permeation events, we were able to statistically investigate the relationship between filter flexibility, conformation and permeation efficiency. The results show a clear correlation between filter flexibility and ion permeation efficiency, indicating that the channel provides more than just a static scaffold to facilitate permeation. In addition, a heterogeneous distribution of ions in the selectivity filter was found during permeation events, indicating that multiple permeation mechanisms concurrently underlie efficient ion permeation.

Unique Mechanism of the Interaction between Honey Bee Toxin TPNQ and rKir1.1 Potassium Channel Explored by Computational Simulations: Insights into the Relative Insensitivity of Channel towards Animal Toxins

BackgroundThe 21-residue compact tertiapin-Q (TPNQ) toxin, a derivative of honey bee toxin tertiapin (TPN), is a potent blocker of inward-rectifier K+ channel subtype, rat Kir1.1 (rKir1.1) channel, and their interaction mechanism remains unclear.Principal FindingsBased on the flexible feature of potassium channel turrets, a good starting rKir1.1 channel structure was modeled for the accessibility of rKir1.1 channel turrets to TPNQ toxin. In combination with experimental alanine scanning mutagenesis data, computational approaches were further used to obtain a reasonable TPNQ toxin-rKir1.1 channel complex structure, which was completely different from the known binding modes between animal toxins and potassium channels. TPNQ toxin mainly adopted its helical domain as the channel-interacting surface together with His12 as the pore-blocking residue. The important Gln13 residue mainly contacted channel residues near the selectivity filter, and Lys20 residue was surrounded by a polar “groove” formed by Arg118, Thr119, Glu123, and Asn124 in the channel turret. On the other hand, four turrets of rKir1.1 channel gathered to form a narrow pore entryway for TPNQ toxin recognition. The Phe146 and Phe148 residues in the channel pore region formed strong hydrophobic protrusions, and produced dominant nonpolar interactions with toxin residues. These specific structure features of rKir1.1 channel vestibule well matched the binding of potent TPNQ toxin, and likely restricted the binding of the classical animal toxins.Conclusions/SignificanceThe TPNQ toxin-rKir1.1 channel complex structure not only revealed their unique interaction mechanism, but also would highlight the diverse animal toxin-potassium channel interactions, and elucidate the relative insensitivity of rKir1.1 channel towards animal toxins.

The interactions of apamin and tetraethylammonium are differentially affected by single mutations in the pore mouth of small conductance calcium-activated potassium (SK) channels

Valine residues in the pore region of SK2 (V366) and SK3 (V520) were replaced by either an alanine or a phenylalanine to evaluate the impact on the interactions with the allosteric blocker apamin. Unlike TEA which showed high sensitivity to phenylalanine mutated channels, the binding affinity of apamin to the phenylalanine mutants was strongly reduced. In addition, currents from phenylalanine mutants were largely resistant to block by apamin. On the other hand, when the valine residue was replaced by an alanine residue, an increase of the binding affinity and the amount of block by apamin was observed for alanine mutated SK2 channels, but not for mutated SK3 channels. Interestingly, the VA mutation reduced the sensitivity to TEA. In silico data confirmed these experimental results. Therefore, such mutations in the pore region of SK channels show that the three-dimensional structure of the SK tetramers can be disorganized in the outer pore region leading to reduced interaction of apamin with its target.

Pore Determinants of KCNQ3 K+ Current Expression

KCNQ3 homomeric channels yield very small macroscopic currents compared with other KCNQ channels or KCNQ2/3 heteromers. Two disparate regions of the channels—the C-terminus and the pore region—have been implicated in governing KCNQ current amplitudes. We previously showed that the C-terminus plays a secondary role compared with the pore region. Here, we confirm the critical role of the pore region in determining KCNQ3 currents. We find that mutations at the 312 position in the pore helix of KCNQ3 (I312E, I312K, and I312R) dramatically decreased KCNQ3 homomeric currents as well as heteromeric KCNQ2/3 currents. Evidence that these mutants were expressed in the heteromers includes shifted TEA sensitivity compared with KCNQ2 homomers. To test for differential membrane protein expression, we performed total internal reflection fluorescence imaging, which revealed only small differences that do not underlie the differences in macroscopic currents. To determine whether this mechanism generalizes to other KCNQ channels, we tested the effects of analogous mutations at the conserved I273 position in KCNQ2, with similar results. Finally, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels to investigate the putative structural mechanism mediating these results. The modeling suggests that the lack of current in I312E, I312K, and I312R KCNQ3 channels is due to pore helix-selectivity filter interactions that lock the selectivity filter in a nonconductive conformation.

Restoration of ion channel function in deafness‐causing KCNQ4 mutants by synthetic channel openers

DFNA2 is a frequent hereditary hearing disorder caused by loss-of-function mutations in the voltage-gated potassium channel KCNQ4 (Kv7.4). KCNQ4 mediates the predominant K(+) conductance, I(K,n) , of auditory outer hair cells (OHCs), and loss of KCNQ4 function leads to degeneration of OHCs resulting in progressive hearing loss. Here we explore the possible recovery of channel activity of mutant KCNQ4 induced by synthetic KCNQ channel openers. Whole cell patch clamp recordings were performed on CHO cells transiently expressing KCNQ4 wild-type (wt) and DFNA2-relevant mutants, and from acutely isolated OHCs. Various known KCNQ channel openers robustly enhanced KCNQ4 currents. The strongest potentiation was observed with a combination of zinc pyrithione plus retigabine. A similar albeit less pronounced current enhancement was observed with native I(K,n) currents in rat OHCs. DFNA2 mutations located in the channel's pore region abolished channel function and these mutant channels were completely unresponsive to channel openers. However, the function of a DFNA2 mutation located in the proximal C-terminus was restored by the combined application of both openers. Co-expression of wt and KCNQ4 pore mutants suppressed currents to barely detectable levels. In this dominant-negative situation, channel openers essentially restored currents back to wt levels, most probably through strong activation of only the small fraction of homomeric wt channels. Our data suggest that by stabilizing the KCNQ4-mediated conductance in OHCs, chemical channel openers can protect against OHC degeneration and progression of hearing loss in DFNA2.

Activation of TRPC Cationic Channels by Mercurial Compounds Confers the Cytotoxicity of Mercury Exposure

Mercury is an established worldwide environmental pollutant with well-known toxicity affecting neurodevelopment in humans, but the molecular basis of cytotoxicity and the detoxification procedure are still unclear. Here we examined the involvement of the canonical transient receptor potential (TRPC) channel in the mercury-induced cytotoxicity and the potential detoxification strategy. Whole-cell and excised patches, Ca(2+) imaging, and site-directed mutagenesis were used to determine the mechanism of action of mercurial compounds on TRPC channels overexpressed in HEK293 cells, and cytotoxicity and preventive effect were investigated in cell culture models using small interfering RNA and pharmacological blockers. Mercury potently activates TRPC4 and TRPC5 channels. The extracellular cysteine residues (C(553) and C(558)) near the channel pore region of TRPC5 are the molecular targets for channel activation by mercury. The sensitivity of mercury to TRPC5 is presumed to be specific because other divalent heavy metal pollutants, such as Cd(2+), Ni(2+), and Zn(2+), had no stimulating effect, and TRPC3, TRPC6, TRPV1, and TRPM2 were resistant to mercurial compounds. The channel activity of TRPC5, as well as TRPC4, induced by mercury, was prevented by 2-aminoethoxydiphenyl borate and modified by a reducing environment. The inhibition of TRPC5 channels by specific TRPC5 pore-blocking antibody or by SKF-96365 alleviated the cytotoxicity, whereas the mercury chelator, meso-2,3-dimercaptosuccinic acid, showed nonselective prevention of cell survival. Silencing of the TRPC5 gene reduced the mercury-induced neuronal damage. These results indicate that mercurial compounds are activators for TRPC5 and TRPC4 channels. Blockade of TRPC channels could be a novel strategy for preventing mercury-induced cytotoxicity and neurodevelopment impairment.

Pore Determinants of KCNQ3 K + Current Expression

Our laboratory has suggested the pore region of KCNQ channels to be implicated in governing current amplitudes, in which large currents are possible by an interaction between a threonine and an isoleucine at the 315 and 312 positions, respectively. Consistent with this, replacement of A315 in KCNQ3 with a threonine or serine increased current amplitudes (Zaika et al., 2008. Biophys J, 95). Here, we find several mutations in KCNQ3 at position 312 (I312V, I312E and I312R) abolished the homomeric current. Co-expression of KCNQ3 I312V and I312E with wild-type (WT) KCNQ2 resulted in smaller currents vs. WT KCNQ2+3 channels, but did not modify channel voltage dependence. Evidence that the I312V and I312E mutants were expressed in the heteromers includes a shifted TEA sensitivity, compared to KCNQ2 homomers. Molecular modeling suggests the lack of current in I312V and I312E KCNQ3 channels to be due to a destabilization of the pore structure. Another lab has suggested the C-terminus KCNQ1-3 channels is critical in determining KCNQ current amplitudes (Schwake et al., 2006. J. Neurosci., 26). Moreover, the crystal structure of the KCNQ4 coiled-coil “D-helix” highlighted three positions (V619, M629 and C643) critical for KCNQ4 channels oligomerization, which are divergent in KCNQ3 (Howard et al., 2007. Neuron, 53). We find H2O2-induced oligomerization of KCNQ4 subunits, reported by native PAGE, to localize to a cysteine located at the end of the D-helix at position 643, at which only KCNQ3 possesses a histidine at the analogous position. As a probe for the role of H646 in KCNQ3 expression, we tested the H646C mutant. However, homomeric or heteromeric channels containing this mutation produced smaller currents, ruling out this divergent residue as underlying low amplitude of KCNQ3 currents. Our results confirm that the pore region is predominant in governing KCNQ channel expression.

P45 Neonatal outcome in late-preterm hypertensive pregnancies – is there an optimal timing for delivery?

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.

1H, 13C and 15N chemical shift assignments for the N-terminal domain of the voltage-gated potassium channel-hERG

The human ether à go-go related gene (hERG) voltage-gated potassium controls the rapid delayed rectifier potassium current (I(ks)) in heart. The N-terminal 135 amino acids (NTD) form a Per-Arnt-Sim (PAS) domain which involves in signal transduction and protein-protein interactions. NTD was shown to be necessary for the regulation of the channel activity through its interaction with the channel pore region of hERG. Mutations in NTD were related to serious heart diseases. We report the (1)H, (13)C and (15)N chemical shift assignments for NTD using 2D and 3D heteronuclear NMR experiments. More than 95% backbone resonance assignments were obtained.

A Bivalent Tarantula Toxin Activates the Capsaicin Receptor, TRPV1, by Targeting the Outer Pore Domain

Toxins have evolved to target regions of membrane ion channels that underlie ligand binding, gating, or ion permeation, and have thus served as invaluable tools for probing channel structure and function. Here, we describe a peptide toxin from the Earth Tiger tarantula that selectively and irreversibly activates the capsaicin- and heat-sensitive channel, TRPV1. This high-avidity interaction derives from a unique tandem repeat structure of the toxin that endows it with an antibody-like bivalency. The "double-knot" toxin traps TRPV1 in the open state by interacting with residues in the presumptive pore-forming region of the channel, highlighting the importance of conformational changes in the outer pore region of TRP channels during activation.

External divalent cations increase anion–cation permeability ratio in glycine receptor channels

The functional role of ligand-gated ion channels in the central nervous system depends on their relative anion-cation permeability. Using standard whole-cell patch clamp measurements and NaCl dilution potential measurements, we explored the effect of external divalent ions on anion-cation selectivity in alpha1-homomeric wild-type glycine receptor channels. We show that increasing external Ca(2+) from 0 to 4 mM resulted in a sigmoidal increase in anion-cation permeability by 37%, reaching a maximum above about 2 mM. Our accurate quantification of this effect required rigorous correction for liquid junction potentials (LJPs) using ion activities, and allowing for an initial offset potential. Failure to do this results in a considerable overestimation of the Ca(2+)-induced increase in anion-cation permeability by almost three-fold at 4 mM external Ca(2+). Calculations of LJPs (using activities)_ were validated by precise agreement with direct experimental measurements. External SO (4) (2-) was found to decrease anion-cation permeability. Single-channel conductance measurements indicated that external Ca(2+) both decreased Na(+) permeability and increased Cl(-) permeability. There was no evidence of Ca(2+) changing channel pore diameter. Theoretical modeling indicates that the effect is not surface charge related. Rather, we propose that, under dilution conditions, the presence of an impermeant Ca(2+) ion in the channel pore region just external to the selectivity filter tends to electrostatically retard outward movement of Na(+) ions and to enhance movement of Cl(-) ions down their energy gradients.

Caesium Permeation Reveals an Unusual Voltage Dependent Gating at the Selectivity Filter of CNGA1 Channels

CNG channels are permeable to alkali monovalent and divalent cations and to small organic cations. Therefore, CNG channels have a low ionic selectivity, attributed to an intrinsic flexibility of the filter region (Laio & Torre, 1999) mediating the coupling between permeation and gating (Gamel & Torre 2000; Holmgren, 2003; Kush, 2004). We have analysed in more detail the permeation of Cs+ in WT CNGA1 channels. In symmetrical Na+ or K+ conditions and in the presence of saturating cGMP concentrations the ratio between the current at +200 and −200 mV I200/I-200 is 1.3. In contrast, in the presence of symmetrical Cs+ I200/I-200 it is about 0.75. Under these conditions, single channel recordings reveal a surprising behaviour: the single channel conductance for Cs+ ions is about 18 pS at −180 mV, but becomes less than 5 pS at voltages larger than 100 mV. The open probability at −180 mV is about 0.2 and becomes close to 1 at +100 mV. When Thr360 is mutated to alanine, the single channel conductance for Cs+ ions is around 15 pS both at +100 and −100 mV. These results confirm the notion that the pore region of CNGA1 channels is highly flexible and that permeating ions modify and control channel gating. These results show also that the pore region of CNGA1 channels acts as a voltage sensor and modifies their conformation in response to changes of the applied field. The molecular mechanisms involved in these rearrangements are likely to consist in reorientation of electric dipoles, such as those of Thr360.

Novel Molecular Determinants in the Pore Region of Sodium Channels Regulate Local Anesthetic Binding

The pore of the Na+ channel is lined by asymmetric loops formed by the linkers between the fifth and sixth transmembrane segments (S5-S6). We investigated the role of the N-terminal portion (SS1) of the S5-S6 linkers in channel gating and local anesthetic (LA) block using site-directed cysteine mutagenesis of the rat skeletal muscle (Na(V)1.4) channel. The mutants examined have variable effects on voltage dependence and kinetics of fast inactivation. Of the cysteine mutants immediately N-terminal to the putative DEKA selectivity filter in four domains, only Q399C in domain I and F1236C in domain III exhibit reduced use-dependent block. These two mutations also markedly accelerated the recovery from use-dependent block. Moreover, F1236C and Q399C significantly decreased the affinity of QX-314 for binding to its channel receptor by 8.5- and 3.3-fold, respectively. Oddly enough, F1236C enhanced stabilization of slow inactivation by both hastening entry into and delaying recovery from slow inactivation states. It is noteworthy that symmetric applications of QX-314 on both external and internal sides of F1236C mutant channels reduced recovery from use-dependent block, indicating an allosteric effect of external QX-314 binding on the recovery of availability of F1236C. These observations suggest that cysteine mutation in the SS1 region, particularly immediate adjacent to the DEKA ring, may lead to a structural rearrangement that alters binding of permanently charged QX-314 to its receptor. The results lend further support for a role for the selectivity filter region as a structural determinant for local anesthetic block.

Biophysical characterization of KCNQ1 P320 mutations linked to long QT syndrome 1

Hereditary long QT syndrome (LQTS) is a cardiovascular disorder characterized by prolongation of the QT interval on the surface ECG and a high risk for arrhythmia-related sudden death. Mutations in a cardiac voltage-gated potassium channel, KCNQ1, account for the most common form of LQTS, LQTS1. The objective of this study was the characterization of a novel KCNQ1 mutation linked to LQTS. Electrophysiological properties and clinical features were determined and compared to characteristics of a different mutation at the same position. Single-strand conformation polymorphism analysis followed by direct sequencing was performed to screen LQTS genes for mutations. A novel missense mutation in the KCNQ1 gene, KCNQ1 P320H, was identified in the index patient presenting with recurrent syncope and aborted sudden death triggered by physical stress and swimming. Electrophysiological analyses of KCNQ1 P320H and the previously reported KCNQ1 P320A mutation indicate that both channels are non-functional and suppress wild type I Ks in a dominant-negative fashion. Based on homology modeling of the KCNQ1 channel pore region, we speculate that the proline residue at position 320 limits flexibility of the outer pore and is required to maintain the functional architecture of the selectivity filter/pore helix arrangement. Our observations on the KCNQ1 P320H mutation are consistent with previous studies indicating that pore mutations in potassium channel α-subunits are associated with more severe electrophysiological and clinical phenotypes than mutations in other regions of these proteins. This study emphasizes the significance of mutation screening for diagnosis, risk-assessment, and mutation-site specific management in LQTS patients.

Evidence that extracellular anions interact with a site outside the CFTR chloride channel pore to modify channel properties

Extracellular anions enter into the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, interacting with binding sites on the pore walls and with other anions inside the pore. There is increasing evidence that extracellular anions may also interact with sites away from the channel pore to influence channel properties. We have used site-directed mutagenesis and patch-clamp recording to identify residues that influence interactions with external anions. Anion interactions were assessed by the ability of extracellular Pt(NO2)42- ions to weaken the pore-blocking effect of intracellular Pt(NO2)42- ions, a long-range ion-ion interaction that does not appear to reflect ion interactions inside the pore. We found that mutations that remove positive charges in the 4th extracellular loop of CFTR (K892Q and R899Q) significantly alter the interaction between extracellular and intracellular Pt(NO2)42- ions. These mutations do not affect unitary Cl- conductance or block of single-channel currents by extracellular Pt(NO2)42- ions, however, suggesting that the mutated residues are not in the channel pore region. These results suggest that extracellular anions can regulate CFTR pore properties by binding to a site outside the pore region, probably by a long-range conformational change. Our findings also point to a novel function of the long 4th extracellular loop of the CFTR protein in sensing and (or) responding to anions in the extracellular solution.

A role for Leu247 residue within transmembrane domain 2 in ginsenoside-mediated alpha7 nicotinic acetylcholine receptor regulation.

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not alpha7, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of alpha7 nAChR induces alterations in channel gating properties and converts alpha7 nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside Rg(3) (Rg(3)) activity against the alpha7 nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to Rg(3). We further characterized Rg(3) regulation of L247T receptors. We found that Rg(3) inhibition of mutant alpha7 nAChR channel currents was reversible and concentration-dependent. Rg(3) inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between Rg(3) and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in Rg(3) interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that Rg(3) forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas Rg(3) localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for Rg(3) at the channel pore.

Functional Consequences Of Disease-associated Mutations In The Pore Region Of Human Cone Photoreceptor CNG Channels

CNGA3 encodes the A-subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel. Mutations in the CNGA3 gene have been associated with achromatopsia, a congenital, autosomal-recessively inherited retinal disorder characterized by lack of color vision, severely reduced visual acuity, photophobia and nystagmus. The aim of this study was the functional characterization of five mutant CNGA3 channels with amino acid substitutions in the pore region (S341P, L363P, G367V, P372S and E376K), which had been identified in achromatopsia patients. Mutant channels were heterologously expressed in HEK293 cells and their functional properties were assessed by calcium imaging and patch-clamp measurements. For patch-clamp recordings mutant CNGA3 was co-expressed with the wild-type B3 subunit present in native channels and transfected cells were incubated at 27°C in order to enhance folding and trafficking of the channel mutants. Furthermore, immunocytochemical experiments were performed after incubation at either 27°C or 37°C to determine the extent of co-localization of mutant channels with the cell membrane.All five pore mutations rendered the channel non-functional in calcium imaging experiments, indicating severely reduced calcium influx through the mutant channel pore. Interestingly, cGMP-induced potassium currents could be recorded from patches containing channels with the mutations S341P, G367V and E376K. Even though macroscopic currents were small compared to wild type channels, these three pore mutants have been shown to possess residual potassium conductivity. While channels with the mutation G367V, P372S or E376K showed normal co-localization with the plasma membrane after incubation at 37°C, reduced surface expression was observed for channel mutants S341P and L363P, suggesting impaired folding and/or trafficking of the mutant proteins.

Fine tuning ICRAC: the interactions of STIM‐1 and Orai

Fine tuning <i>I</i>_CRAC: the interactions of STIM‐1 and Orai

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 Ba 2+ 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.

Audioprofile-directed screening identifies novel mutations in KCNQ4 causing hearing loss at the DFNA2 locus

PurposeGene identification in small families segregating autosomal dominant sensorineural hearing loss presents a significant challenge. To address this challenge, we have developed a machine learning-based software tool, AudioGene v2.0, to prioritize candidate genes for mutation screening based on audioprofiling. MethodsWe analyzed audiometric data from a cohort of American families with high-frequency autosomal dominant sensorineural hearing loss. Those families predicted to have a DFNA2 audioprofile by AudioGene v2.0 were screened for mutations in the KCNQ4 gene. ResultsTwo novel missense mutations and a stop mutation were detected in three American families predicted to have DFNA2-related deafness for a positive predictive value of 6.3%. The false negative rate was 0%. The missense mutations were located in the channel pore region and the stop mutation was in transmembrane domain S5. The latter is the first DFNA2-causing stop mutation reported in KCNQ4. ConclusionsOur data suggest that the N-terminal end of the P-loop is crucial in maintaining the integrity of the KCNQ4 channel pore and AudioGene audioprofile analysis can effectively prioritize genes for mutation screening in small families segregating high-frequency autosomal dominant sensorineural hearing loss. AudioGene software will be made freely available to clinicians and researchers once it has been fully validated.