Voltage Sensitivity Of Channels Research Articles

Epidermal growth factor modulates voltage sensitivity of slow sodium channels

The objective of this study was to determine the mechanisms underlying excitation of the nociceptive neuronal membrane under the influence of growth factors. Each ionic channel or its gate structure is known to have a specific function during the primary sensory encoding. For example, adaptation is a result of activation of slow sodium channels or it is caused by complicated behavior of the activation gate structure of sodium channels (1, 2). Receptormediated modu� lation of the activation gate structure of slow sodium channels also reduces the frequency of recurrent responses of the nociceptive neuronal membrane (3). This antinociceptive effect may be caused by the epi� dermal growth factor (EGF). Our study demonstrates that precisely this growth factor modulates the voltage sensitivity of slow tetrodotoxininsensitive sodium channels (Na v 1.8), which are responsible for encoding the pain signal (4, 5). Growth factors are polypeptides constituting a group of trophic regulatory substances. EGF is the main polypeptide stimulating growth of endometrium cells (6). EGF polypeptide with a molecular weight of about 6 kDa is composed of 53 amino acid residues and has an effect of a strong mitogen on various cells of the endo�, ecto�, and mesodermal origin. Low EGF concentrations (above 2 ng/mL) were determined in blood, cerebrospinal fluid, milk, saliva, gastric and pancreatic juices (7).

Genotype–phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor of K v 7.2 potassium channel subunits

Mutations in the K(V)7.2 gene encoding for voltage-dependent K(+) channel subunits cause neonatal epilepsies with wide phenotypic heterogeneity. Two mutations affecting the same positively charged residue in the S4 domain of K(V)7.2 have been found in children affected with benign familial neonatal seizures (R213W mutation) or with neonatal epileptic encephalopathy with severe pharmacoresistant seizures and neurocognitive delay, suppression-burst pattern at EEG, and distinct neuroradiological features (R213Q mutation). To examine the molecular basis for this strikingly different phenotype, we studied the functional characteristics of mutant channels by using electrophysiological techniques, computational modeling, and homology modeling. Functional studies revealed that, in homomeric or heteromeric configuration with K(V)7.2 and/or K(V)7.3 subunits, both mutations markedly destabilized the open state, causing a dramatic decrease in channel voltage sensitivity. These functional changes were (i) more pronounced for channels incorporating R213Q- than R213W-carrying K(V)7.2 subunits; (ii) proportional to the number of mutant subunits incorporated; and (iii) fully restored by the neuronal K(v)7 activator retigabine. Homology modeling confirmed a critical role for the R213 residue in stabilizing the activated voltage sensor configuration. Modeling experiments in CA1 hippocampal pyramidal cells revealed that both mutations increased cell firing frequency, with the R213Q mutation prompting more dramatic functional changes compared with the R213W mutation. These results suggest that the clinical disease severity may be related to the extent of the mutation-induced functional K(+) channel impairment, and set the preclinical basis for the potential use of K(v)7 openers as a targeted anticonvulsant therapy to improve developmental outcome in neonates with K(V)7.2 encephalopathy.

Voltage-Dependence in Thermo-Voltage Sensitive Channel TRPV1. A Delocalized Voltage Sensor?

Every organism needs molecular antennae in order to sense the environmental temperature. In mammals, these antennae are a set of ion channels called thermo Transient Receptor Potential (thermoTRP). TRPV1 is a heat activated channel that belongs to TRP family, with an activation threshold around 42 °C. Experimental evidences show that TRPV1 posses a similar structural architecture to voltage-dependent K+ channels (Kv). Nevertheless, TRPV1 has a weakly voltage dependence (number of apparent gating charges, z = 0.83 e0) and in contrast with the numerous positively charged residues contained in the S4 of Kv channels (∼12 e0 gating charges/channel), it has only one arginine (R557) located in the fourth transmembrane segment (S4). However, the molecular determinant that confers the voltage sensitivity to this channel remains elusive. using site-directed mutagenesis we have neutralized several charged amino acid (positives and negatives) along to the channel. The electrophysiological studies (macro-patch in X. laevis oocytes), surprisingly reveals that the uncharged S4 TRPV1 channels still conserve the same voltage dependence as wt channels. Furthermore, neutralizations in other transmembrane segments or even neutralizations on the pore region do not affect the voltage sensitivity of TRPV1 channels. Despite the weak voltage dependence of TRPV1 channel, the membrane voltage has a huge impact over the temperature activation; changing the activation enthalpy and the ionic current kinetics. At hyperpolarizing potentials the temperature activation shows a delay resembling the Cole-Moore shift. Furthermore, at depolarizing potential the channel kinetics speeds up, revealing an allosteric connection between voltage and thermal stimuli. This work was supported by Fondecyt grant 1110430 to RL, Fondecyt 1120802 to CG and ACT 10224 to CG. CINV is a Scientific Millennium Institute.

A Theoretical Model for Calculating Voltage Sensitivity of Ion Channels and the Application on Kv1.2 Potassium Channel

Voltage sensing confers conversion of a change in membrane potential to signaling activities underlying the physiological processes. For an ion channel, voltage sensitivity is usually experimentally measured by fitting electrophysiological data to Boltzmann distributions. In our study, a two-state model of the ion channel and equilibrium statistical mechanics principle were used to test the hypothesis of empirically calculating the overall voltage sensitivity of an ion channel on the basis of its closed and open conformations, and determine the contribution of individual residues to the voltage sensing. We examined the theoretical paradigm by performing experimental measurements with Kv1.2 channel and a series of mutants. The correlation between the calculated values and the experimental values is at respective level, R2 = 0.73. Our report therefore provides in silico prediction of key conformations and has identified additional residues critical for voltage sensing.

A mutation in a CLC anion channel alters serotonergic neuronal activity in C. elegans

Egg‐laying in C. elegans is controlled by the HSN neurons, which release serotonin to activate egg‐laying muscles. The dominant egl‐42(n995) mutation prevents serotonin release and inhibits egg‐laying. Intracellular Ca2+ imaging demonstrated that HSN activity is reduced in egl‐42 mutants. Whole genome sequencing revealed that egl‐42(n995) is a point mutation in the clh‐3 gene, which encodes the CLC anion channel splice variants CLH‐3a and CLH‐3b. Overexpression of both wild‐type and mutant clh‐3 phenocopies the egl‐42(n995) egg‐laying defect, while clh‐3 deletion leads to hyperactive egg‐laying. The n995 allele is a valine to alanine mutation that is located in membrane helix P, which contributes to the monomer‐monomer interface in CLC homodimers. This residue is highly conserved in all human CLC proteins except CLC‐Ka and CLC‐Kb. CLH‐3a and CLH‐3b mutant channels exhibit increased current amplitude, a strongly depolarized activation voltage, and more rapid voltage dependent activation kinetics. CLH‐3a and CLH‐3b mutants also exhibit loss of pre‐potentiation behavior and greatly reduced sensitivity to inhibition by GCK‐3 kinase, respectively. Our results demonstrate 1) that CLC helix P plays an important role in controlling channel voltage sensitivity and 2) that clh‐3 encoded channels likely regulate HSN membrane potential and hence Ca2+ entry and neuronal excitability.

Angiotensin II Type 1 Receptor Mediates Angiotensin II Inhibition on BKCa Channels

The renin-angiotensin system (RAS) is a critical regulator of sodium balance, extracellular fluid volume, vascular resistance, and, ultimately, arterial blood pressure. The key RAS molecule angiotensin II type 1 receptor (AT1R), and another major determinant of vascular tone, the large conductance calcium-activated potassium (BKCa) channel, are both highly expressed in renal arterial smooth muscle cells (SMCs). Our previous studies in expression systems revealed a physical association between AT1R and BKCa, and that AT1R association modified BKCa channel voltage sensitivity. However, the effect of Ang II on BKCa channels in renal arterial SMCs needs to be defined. Furthermore, whether AT1R association is critical for the alteration of channel activity in response to Ang II, and whether the coupling changes BKCa reactivity to specific inhibitors also remain unknown. Our present studies in rat renal arterial SMCs show that application of 100 nM Iberiotoxin (IbTx, a specific BKCa channel blocker) inhibits whole-cell BKCa currents by 63.0±12.5% (n=5). IbTx-sensitive BKCa currents were reduced by 44.4±8.7% (n=3) in response to 1 μM Ang II treatment. In AT1R-IRES-BKCa-transfected HEK293T cells, extracellular application of 1 μM Ang II also suppressed whole-cell BKCa currents by 19.7±0.3% (n=3), while this treatment made no significant changes in cells expressing only BKCa (n=6). IbTx reduced the whole-cell currents by 37.2±1.9% (n=4) in BKCa-transfected HEK293T cells and 37.7±12.5% (n=5) in AT1R-IRES-BKCa-transfected cells. Paxilline treatment (100 nM) produced a 66.8±8.0% (n=3) reduction of BKCa whole-cell currents in BKCa-transfected HEK293T cells and 65.8±9.2% (n=3) reduction in AT1R-IRES-BKCa-transfected cells. These results demonstrate that Ang II inhibits BKCa channel activity in renal arterial SMCs and suggest that AT1R serves as the mediator of the effect. However, the interaction between AT1R and BKCa does not change channel reactivity towards specific channel inhibitors. Supported by NIH.

Muscle weakness in myotonic dystrophy associated with misregulated splicing and altered gating of CaV1.1 calcium channel

Myotonic dystrophy type 1 and type 2 (DM1 and DM2) are genetic diseases in which mutant transcripts containing expanded CUG or CCUG repeats cause cellular dysfunction by altering the processing or metabolism of specific mRNAs and miRNAs. The toxic effects of mutant RNA are mediated partly through effects on proteins that regulate alternative splicing. Here we show that alternative splicing of exon 29 (E29) of Ca(V)1.1, a calcium channel that controls skeletal muscle excitation-contraction coupling, is markedly repressed in DM1 and DM2. The extent of E29 skipping correlated with severity of weakness in tibialis anterior muscle of DM1 patients. Two splicing factors previously implicated in DM1, MBNL1 and CUGBP1, participated in the regulation of E29 splicing. In muscle fibers of wild-type mice, the Ca(V)1.1 channel conductance and voltage sensitivity were increased by splice-shifting oligonucleotides that induce E29 skipping. In contrast to human DM1, expression of CUG-expanded RNA caused only a modest increase in E29 skipping in mice. However, forced skipping of E29 in these mice, to levels approaching those observed in human DM1, aggravated the muscle pathology as evidenced by increased central nucleation. Together, these results indicate that DM-associated splicing defects alter Ca(V)1.1 function, with potential for exacerbation of myopathy.

Surface charge impact in low-magnesium model of seizure in rat hippocampus

Putative mechanisms of induction and maintenance of seizure-like activity (SLA) in the low Mg(2+) model of seizures are: facilitation of NMDA receptors and decreased surface charge screening near voltage-gated channels. We have estimated the role of such screening in the early stages of SLA development at both physiological and room temperatures. External Ca(2+) and Mg(2+) promote a depolarization shift of the sodium channel voltage sensitivity; when examined in hippocampal pyramidal neurons, the effect of Ca(2+) was 1.4 times stronger than of Mg(2+). Removing Mg(2+) from the extracellular solution containing 2 mM Ca(2+) induced recurrent SLA in hippocampal CA1 pyramidal layer in 67% of slices. Reduction of [Ca(2+)](o) to 1 mM resulted in 100% appearance of recurrent SLA or continuous SLA. Both delay before seizure activity and the inter-SLA time were significantly reduced. Characteristics of seizures evoked in low Mg(2+)/1 mM Ca(2+)/3.5 K(+) were similar to those obtained in low Mg(2+)/2 Ca(2+)/5mM K(+), suggesting that reduction of [Ca(2+)](o) to 1 mM is identical to the increase in [K(+)](o) to 5 mM in terms of changes in cellular excitability and seizure threshold. An increase of [Ca(2+)](o) to 3 mM completely abolished SLA generation even in the presence of 5 mM [K(+)](o). A large variation in the ability of [Ca(2+)](o) to stop epileptic discharges in initial stage of SLA was found. Our results indicate that surface charge of the neuronal membrane plays a crucial role in the initiation of low Mg(2+)-induced seizures. Furthermore, our study suggests that Ca(2+) and Mg(2+), through screening of surface charge, have important anti-seizure and antiepileptic properties.

Chloride Ions in the Pore of Glycine and GABA Channels Shape the Time Course and Voltage Dependence of Agonist Currents

In the vertebrate CNS, fast synaptic inhibition is mediated by GABA and glycine receptors. We recently reported that the time course of these synaptic currents is slower when intracellular chloride is high. Here we extend these findings to measure the effects of both extracellular and intracellular chloride on the deactivation of glycine and GABA currents at both negative and positive holding potentials. Currents were elicited by fast agonist application to outside-out patches from HEK-293 cells expressing rat glycine or GABA receptors. The slowing effect of high extracellular chloride on current decay was detectable only in low intracellular chloride (4 mm). Our main finding is that glycine and GABA receptors "sense" chloride concentrations because of interactions between the M2 pore-lining domain and the permeating ions. This hypothesis is supported by the observation that the sensitivity of channel gating to intracellular chloride is abolished if the channel is engineered to become cation selective or if positive charges in the external pore vestibule are eliminated by mutagenesis. The appropriate interaction between permeating ions and channel pore is also necessary to maintain the channel voltage sensitivity of gating, which prolongs current decay at depolarized potentials. Voltage dependence is abolished by the same mutations that suppress the effect of intracellular chloride and also by replacing chloride with another permeant ion, thiocyanate. These observations suggest that permeant chloride affects gating by a foot-in-the-door effect, binding to a channel site with asymmetrical access from the intracellular and extracellular sides of the membrane.

Common Molecular Determinants of Tarantula Huwentoxin-IV Inhibition of Na+ Channel Voltage Sensors in Domains II and IV

The voltage sensors of domains II and IV of sodium channels are important determinants of activation and inactivation, respectively. Animal toxins that alter electrophysiological excitability of muscles and neurons often modify sodium channel activation by selectively interacting with domain II and inactivation by selectively interacting with domain IV. This suggests that there may be substantial differences between the toxin-binding sites in these two important domains. Here we explore the ability of the tarantula huwentoxin-IV (HWTX-IV) to inhibit the activity of the domain II and IV voltage sensors. HWTX-IV is specific for domain II, and we identify five residues in the S1-S2 (Glu-753) and S3-S4 (Glu-811, Leu-814, Asp-816, and Glu-818) regions of domain II that are crucial for inhibition of activation by HWTX-IV. These data indicate that a single residue in the S3-S4 linker (Glu-818 in hNav1.7) is crucial for allowing HWTX-IV to interact with the other key residues and trap the voltage sensor in the closed configuration. Mutagenesis analysis indicates that the five corresponding residues in domain IV are all critical for endowing HWTX-IV with the ability to inhibit fast inactivation. Our data suggest that the toxin-binding motif in domain II is conserved in domain IV. Increasing our understanding of the molecular determinants of toxin interactions with voltage-gated sodium channels may permit development of enhanced isoform-specific voltage-gating modifiers.

Palmitoylation of the S0-S1 Linker Regulates Cell Surface Expression of Voltage- and Calcium-activated Potassium (BK) Channels

S-Palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium- and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by ∼55% in the C53:54:56A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.

Voltage-Dependent Gating in a “Voltage Sensor-Less” Ion Channel

Author SummaryIon channels are proteins that regulate the transfer of ions across the cell membrane. The ions travel via a pore formed by the different subunits that constitute the channels, and this pore can be gated by changes in the electrical field across cell membranes. The canonical mechanism underlying voltage dependence of gating relies upon a widely conserved structural motif called the voltage sensor, which undergoes conformational changes when charged amino acids within the motif respond to voltage and consequently affect the opening of the ion channel pore. In the present study, we have identified a non-canonical mechanism that surprisingly generates voltage-dependent changes in the activity of a ligand-gated ion channel that has no voltage sensor. Our observations suggest that ions flowing through the ion channel pore can significantly affect channel activity, and we suggest that voltage-dependent changes in ion distribution in the “cavity site” of the channel can influence opening and closing of the channel independent of canonical voltage sensors.

An Intrinsic Neuronal Oscillator Underlies Dopaminergic Neuron Bursting

Dopaminergic neurons of the ventral midbrain fire high-frequency bursts when animals are presented with unexpected rewards, or stimuli that predict reward. To identify the afferents that can initiate bursting and establish therapeutic strategies for diseases affected by altered bursting, a mechanistic understanding of bursting is essential. Our results show that bursting is initiated by a specific interaction between the voltage sensitivity of NMDA receptors and voltage-gated ion channels that results in the activation of an intrinsic, action potential-independent, high-frequency membrane potential oscillation. We further show that the NMDA receptor is uniquely suited for this because of the rapid kinetics and voltage dependence imparted to it by Mg(2+) ion block and unblock. This mechanism explains the discrete nature of bursting in dopaminergic cells and demonstrates how synaptic signals may be reshaped by local intrinsic properties of a neuron before influencing action potential generation.

Phosphatidylinositol 4,5-bisphosphate and loss of PLCγ activity inhibit TRPM channels required for oscillatory Ca2+ signaling

The Caenorhabditis elegans intestinal epithelium generates rhythmic inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) oscillations that control muscle contractions required for defecation. Two highly Ca(2+)-selective transient receptor potential (TRP) melastatin (TRPM) channels, GON-2 and GTL-1, function with PLCgamma in a common signaling pathway that regulates IP(3)-dependent intracellular Ca(2+) release. A second PLC, PLCbeta, is also required for IP(3)-dependent Ca(2+) oscillations, but functions in an independent signaling mechanism. PLCgamma generates IP(3) that regulates IP(3) receptor activity. We demonstrate here that PLCgamma via hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) also regulates GON-2/GTL-1 function. Knockdown of PLCgamma but not PLCbeta activity by RNA interference (RNAi) inhibits channel activity approximately 80%. Inhibition is fully reversed by agents that deplete PIP(2) levels. PIP(2) added to the patch pipette has no effect on channel activity in PLCgamma RNAi cells. However, in control cells, 10 microM PIP(2) inhibits whole cell current approximately 80%. Channel inhibition by phospholipids is selective for PIP(2) with an IC(50) value of 2.6 microM. Elevated PIP(2) levels have no effect on channel voltage and Ca(2+) sensitivity and likely inhibit by reducing channel open probability, single-channel conductance, and/or trafficking. We conclude that hydrolysis of PIP(2) by PLCgamma functions in the activation of both the IP(3) receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell Ca(2+) influx pathway. Calcium influx through the channel feedback regulates its activity and likely functions to modulate IP(3) receptor function. PIP(2)-dependent regulation of GON-2/GTL-1 may provide a mechanism to coordinate plasma membrane Ca(2+) influx with PLCgamma and IP(3) receptor activity as well as intracellular Ca(2+) store depletion.

Electromagnetic field (EMF) effects on channel activity of nanopore OmpF protein

In this study, the effects of nonionizing electromagnetic fields (EMF; 925 MHz) on the OmpF porin channel have been characterized at the single-channel level. Channel activity was recorded in real time by the voltage clamp method. Our results showed an increase in the frequency of channel gating and voltage sensitivity. The effects of EMF lasted for several milliseconds after the field source was terminated. However, the conductance levels of channels did not change significantly. Thermal effects of EMF on single-channel properties are a possible cause, based on theoretical evaluation of results that were comparable to those seen in conventional experiments at different temperatures. We conclude that EMF affects both the dynamics and conformation of the channel, either directly by affecting critical amino acid side-chain arrangement, or indirectly, via the electrolyte or the lipid membrane.

Enhancement of Voltage Sensitivity of a cGMP-gated Channel

Activity of cyclic nucleotide-gated (CNG) cation channels underlies signal transduction in vertebrate visual receptors. These channels must be primarily activated by the binding of cGMP so that the activity of these highly specialized receptor channels be controlled by ligands in a finely graded manner required for transducing sensory stimuli of varying intensity. Significant voltage sensitivity of the channels would generate voltage-driven positive feedback and thus reduce the signal-transduction sensitivity. Indeed, the CNGA1 channel is only modestly voltage sensitive in low cGMP concentrations, and the voltage sensitivity vanishes with increasing cGMP concentration. We have found that loosening the attachment of the selectivity filter to the surrounding “pore shell” dramatically increases the channel's voltage sensitivity, which is independent of the positively charged residues in S4. Thus, proper attachment of the selectivity filter is essential to avoid significant, adverse voltage sensitivity in these channels.

The Positive Effect Of STREX On BK Channels

The large conductance voltage and calcium sensitive potassium channel (BK) is encoded by a single gene KCNMA1. The inclusion of a stress regulated exon (STREX) at a splice site (C2) in the intracellular carboxyl (C) terminus of BK confers differing properties to the channel. STREX insertion generates a putative polybasic region. In a number of other channels, polybasic regions are suggested to interact with negatively charged phospholipids, such as phosphoinositides, to control ion channel gating and membrane targeting. We hypothesised that the polybasic region including STREX may serve as a membrane targeting domain of the STREX BK channel C-terminus. To test this, a GFP-tagged carboxyl terminal construct spanning the S6 transmembrane domain to the COOH end region of the intracellular carboxyl terminus was constructed (S6-COOH-STX) and transiently transfected into HEK293cells. The construct localised at the plasma membrane and this was abolished when the STREX insert was deleted. To test whether the polybasic region is important for plasma membrane targeting, two approaches were taken: Firstly, site directed mutagenesis to change selected positive residues into neutral (alanine) residues, also abolished membrane targeting of S6-COOH-STX to the plasma membrane. Secondly, to discern whether the polybasic region may interact with negatively charged phosphoinositides at the plasma membrane, the S6-COOH-STX construct was co-transfected with a 5′phosphatase, IPP. Cells co-expressing IPP displayed significantly reduced plasma membrane targeting of S6-COOH-STX, however a phosphatase null mutant of IPP did not effect plasma membrane expression. These data suggest that the polybasic region generated by inclusion of the STREX insert is an important determinant of STREX domain interaction with the plasma membrane. The functional role of the polybasic region and the interaction of phospholipids on BK channel calcium and voltage sensitivity were elucidated using patch clamp electrophysiology and high throughput dyes.

Trifluoroethanol reveals helical propensity at analogous positions in cytoplasmic domains of three connexins

Cytoplasmic domains of gap junction proteins (connexins) are involved in channel gating, voltage and pH sensitivity, and contain binding sites for partner proteins. However, their secondary structure is incompletely characterized and comparisons among the connexins is totally lacking. Circular dichroism (CD) was used to study the conformational properties of synthetic peptides corresponding to the highly divergent amino acid sequences of cytoplasmic domains of connexin (Cx)32, Cx36, and Cx43. We report that whereas peptides were largely unstructured in aqueous buffer, certain peptides in 30% trifluoroethanol (TFE) showed considerable helical content. These structured peptides correspond to analogous regions in each of the three connexin cytoplasmic domains. This first comparative study of conformational properties of connexin cytoplasmic domains reveals protein domains that may play similar roles in channel function and protein-protein interactions.

Non-linear intramolecular interactions and voltage sensitivity of a KV1 family potassium channel fromPolyorchis penicillatus(Eschscholtz 1829)

Voltage sensitivity of voltage-gated potassium channels (VKCs) is a primary factor in shaping action potentials in excitable cells. Variation in the amino acid sequence of the channel proteins is responsible for differences in the voltage range over which the channel opens. Thus, understanding how changes in voltage sensitivity are effected by changes in channel protein sequence illuminates the functional evolution of excitability. The K(V)1-family channel jShak1, from the jellyfish Polyorchis penicillatus, differs from most other K(V)1 channels in ways that are useful for studying the problem of how voltage sensitivity is related to channel sequence. We assessed the contributions of changes in sequence of the S4, voltage sensing, helix and changes in one asparagine residue in the S2 helix, to the relative stability of the open and closed states of the channel. Mutation of the neutral S2 residue (Asn227) to glutamate stabilized the open conformation of the channel. Different modifications of charge and length in S4 favoured either the closed conformation or the open conformation. The interactions between pairs of mutations revealed that some of the S4 mutations alter the conformation of the voltage-sensing domain such that the S4 helix is constrained to be closer to the S2 helix than in the wild-type conformation. These results, taken in conjunction with three-dimensional models of the channel, identify intra-molecular interactions that control the balance between open and closed states. These interactions are likely to be relevant to understanding the functional characteristics of members of this channel family from other organisms.

Voltage Gating at the Selectivity Filter of the Ca2+ Release-activated Ca2+ Channel Induced by Mutation of the Orai1 Protein

The Ca(2+) release-activated Ca(2+) (CRAC) channel is a plasma membrane (PM) channel that is uniquely activated when free Ca(2+) level in the endoplasmic reticulum (ER) is substantially reduced. Several small interfering RNA screens identified two membrane proteins, Orai1 and STIM1, to be essential for the CRAC channel function. STIM1 appears to function in the PM and as the Ca(2+) sensor in the ER. Orai1 is forming the pore of the CRAC channel. Despite the recent breakthroughs, a mechanistic understanding of the CRAC channel gating is still lacking. Here we reveal new insights on the structure-function relationship of STIM1 and Orai1. Our data suggest that the cytoplasmic coiled-coil region of STIM1 provides structural means for coupling of the ER membrane to the PM to activate the CRAC channel. We mutated two hydrophobic residues in this region to proline (L286P/L292P) to introduce a kink in the first alpha-helix of the coiled-coil domain. This STIM1 mutant caused a dramatic inhibition of the CRAC channel gating compared with the wild type. Structure-function analysis of the Orai1 protein revealed the presence of intrinsic voltage gating of the CRAC channel. A mutation of Orai1 (V102I) close to the selectivity filter modified CRAC channel voltage sensitivity. Expression of the Orai1(V102I) mutant resulted in slow voltage gating of the CRAC channel by negative potentials. The results revealed that the alteration of Val(102) develops voltage gating in the CRAC channel. Our data strongly suggest the presence of a novel voltage gating mechanism at the selectivity filter of the CRAC channel.