Gating Polarity Research Articles

간극결합채널의 아미노말단이 채널개폐에 미치는 영향

간극결합은 이웃하는 두 세포 사이에 형성된 이온채널이며 또한 단일세포막에서도 작용한다. 간극결합채널을 형성하는 아미노 말단의 10번째 아미노산 잔기 부위까지가 개폐극성(gating polarity)과 전류-전압관계에 영향을 미친다. 정상적인 Cx32 채널은 음성의 개폐극성과 내향적인 정류현상을 보이는 반면, 음성전하를 띠는 aspartate로 치환된 T8D 채널은 반대의 개폐극성과 직선의 정류현상을 보인다. 이러한 개폐극성과 정류현상의 변화가 전하 자체에 의한 것인지 아니면 아미노 말단의 구조적인 변화에 의한 것인지는 아직 불명확하다. 이러한 문제점을 규명하기 위하여 아미노 말단의 8번째 아미노산 잔기를 cysteine기로 치환시킨 T8C 채널을 만들어 substituted-cysteine accessibility method (SCAM) 방법으로 이 채널의 생물리학적 특성을 조사하고자 하였다. T8C 채널은 정상적인 Cx32 채널처럼 음성의 개폐극성과 내향적인 정류현상을 보였으며, cysteine기로 치환이 정상적인 Cx32 채널의 원래 구조를 변화시키지 않았다는 것을 의미한다. 본 연구에서는 이런 전하효과를 규명하기 위하여 음성 전하를 갖는 MTSES-와 양성전하를 갖는 MTSET+를 사용하였다. MTSES-를 처리하면 T8C 채널은 T8D 채널의 특성처럼 양성의 개폐극성과 직선의 정류현상을 보였다. 그러나 양성전하를 갖는 MTSET+를 처리한 경우에는 T8C 채널은 본래의 특성을 그대로 유지하였다. 작은 분자의 MTS에 의해서 부여된 전하가 아미노 말단의 구조적인 변화를 초래하지는 않을 것으로 생각된다. 따라서 반대의 전하를 띠는 MTSES-와 MTSET+가 서로 상반대는 영향을 미치는 것으로 보아 본 연구에서 관찰된 개폐극성과 전류-전압의 변화는 아미노말단의 구조적인 변화라기보다는 MTS에 의해서 부여된 전하 자체에 기인한다고 할 수 있다. 또한 MTS가 아미노말단의 8번째 부위에 접근하여 반응을 일으킬 수 있다는 결과는 간극결합채널의 아미노말단이 채널의 통로(pore)를 형성한다는 가설을 뒷받침한다. Gap junction is an ion channel forming between adjacent cells. It also acts as a membrane channel like sodium or potassium channels in a single cell. The amino acid residues up to the <TEX>$10^{th}$</TEX> position in the amino (N)-terminus of gap junction hemichannel affect gating polarity as well as current-voltage (I-V) relation. While wild-type Cx32 channel shows negative gating polarity and inwardly rectifying I-V relation, T8D channel in which threonine residue at <TEX>$8^{th}$</TEX> position is replaced with negatively charged aspartate residue shows reverse gating polarity and linear I-V relation. It is still unclear whether these changes are resulted from the charge effect or the conformational change of the N-terminus. To clarify this issue, we made a mutant channel harboring cysteine residue at the <TEX>$8^{th}$</TEX> position (T8C) and characterized its biophysical properties using substituted-cysteine accessibility method (SCAM). T8C channel shows negative gating polarity and inwardly rectifying I-V relation as wild-type channel does. This result indicates that the substitution of cysteine residue dose not perturb the original conformation of wild-type channel. To elucidate the charge effect two types of methaenthiosulfonate (MTS) reagents (negatively charged <TEX>$MTSES^-$</TEX> and positively charged <TEX>$MTSET^+$</TEX>) were used. When <TEX>$MTSES^-$</TEX> was applied, T8C channel behaved as T8D channel, showing positive gating polarity and linear I-V relation. This result indicates that the addition of a negative charge changes the biophysical properties of T8C channel. However, positively charged <TEX>$MTSET^+$</TEX> maintained the main features of T8C channel as expected. It is likely that the addition of a charge by small MTS reagents does not distort the conformation of the N-terminus. Therefore, the opposite effects of <TEX>$MTSES^-$</TEX> and <TEX>$MTSETT^+$</TEX> on T8C channel suggest that the addition of a charge itself rather than the conformational change of the N-terminus changes gating polarity and I-V relation. Furthermore, the accessibility of MTS reagents to amino acid residues at the <TEX>$8^{th}$</TEX> position supports the idea that the N-terminus of gap junction channel forms or lies in the aqueous pore.

Ion Permeation through a Voltage- Sensitive Gating Pore in Brain Sodium Channels Having Voltage Sensor Mutations

Voltage-gated sodium channels activate in response to depolarization, but it is unknown whether the voltage-sensing arginines in their S4 segments pivot across the lipid bilayer as voltage sensor paddles or move through the protein in a gating pore. Here we report that mutation of pairs of arginine gating charges to glutamine induces cation permeation through a gating pore in domain II of the Na(V)1.2a channel. Mutation of R850 and R853 induces a K(+)-selective inward cationic current in the resting state that is blocked by activation. Remarkably, mutation of R853 and R856 causes an outward cationic current with the opposite gating polarity. These results support a model in which the IIS4 gating charges move through a narrow constriction in a gating pore in the sodium channel protein during gating. Paired substitutions of glutamine allow cation movement through the constriction when appropriately positioned by the gating movements of the S4 segment.

Inversion of both gating polarity and CO2 sensitivity of voltage gating with D3N mutation of Cx50

The effect of CO(2)-induced acidification on transjunctional voltage (V(j)) gating was studied by dual voltage-clamp in oocytes expressing mouse connexin 50 (Cx50) or a Cx50 mutant (Cx50-D3N), in which the third residue, aspartate (D), was mutated to asparagine (N). This mutation inverted the gating polarity of Cx50 from positive to negative. CO(2) application greatly decreased the V(j) sensitivity of Cx50 channels, and increased that of Cx50-D3N channels. CO(2) also affected the kinetics of V(j) dependent inactivation of junctional current (I(j)), decreasing the gating speed of Cx50 channels and increasing that of Cx50-D3N channels. In addition, the D3N mutation increased the CO(2) sensitivity of chemical gating such that even CO(2) concentrations as low as 2.5% significantly lowered junctional conductance (G(j)). With Cx50 channels G(j) dropped by 78% with a drop in intracellular pH (pH(i)) to 6.83, whereas with Cx50-D3N channels G(j) dropped by 95% with a drop in pH(i) to just 7.19. We have previously hypothesized that the way in which V(j) gating reacts to CO(2) might be related to connexin's gating polarity. This hypothesis is confirmed here by evidence that the D3N mutation inverts the gating polarity as well as the effect of CO(2) on V(j) gating sensitivity and speed.

Opposite Cx32 and Cx26 Voltage-Gating Response to CO2 Reflects Opposite Voltage-Gating Polarity

Previous studies have shown that the V(j)-dependent gating behavior of gap junction channels is altered by CO(2) exposure. V(j)-dependent channel closure is increased by CO(2) in some connexin channels and decreased in others. Since the former type of channels gate on the relatively negative side by V(j) (negative gaters) and the latter at the positive side (positive gaters), it has been hypothesized that gating polarity determines the way CO(2) affects V(j) closure. To test this hypothesis, we have studied the CO(2)-mediated changes in V(j) gating in channels made of Cx32, Cx26, or a Cx32 mutant (Cx32-N2D) in which asparagine (N) at position 2 was replaced with aspartate (D). With exposure to CO(2), Cx32 channels (negative gaters) show increased V(j)-dependent closure, whereas Cx26 channels (positive gaters) respond in the opposite way to V(j). Additionally, Cx32-N2D channels (positive gaters) show decreased V(j) closure with exposure to CO(2). The reciprocal Cx26 mutant, Cx26-D2N (negative gater), could not be tested because it did not express functional homotypic channels. The data support the hypothesis that polarity of fast V(j) gating determines whether CO(2) increases or decreases the V(j) dependent closure of gap junction channels.

Determinants of Gating Polarity of a Connexin 32 Hemichannel

There is good evidence supporting the view that the transjunctional voltage sensor (V j-sensor) of Cx32 and other Group 1 connexins is contained within a segment of the N-terminus that contributes to the formation of the channel pore. We have shown that the addition of negatively charged amino acid residues at several positions within the first 10 amino acid residues reverses the polarity of V j-gating and proposed that channel closure is initiated by the inward movement of this region. Here, we report that positive charge substitutions of the 2nd, 5th, and 8th residues maintain the negative polarity of V j-gating. These data are consistent with the original gating model. Surprisingly, some channels containing combinations of positive and/or negative charges at the 2nd and 5th positions display bipolar V j-gating. The appearance of bipolar gating does not correlate with relative orientation of charges at this position. However, the voltage sensitivity of bipolar channels correlates with the sign of the charge at the 2nd residue, suggesting that charges at this position may have a larger role in determining gating polarity. Taken together with previous findings, the results suggest that the polarity V j-gating is not determined by the sign of the charge lying closest to the cytoplasmic entry of the channel, nor is it likely to result from the reorientation of an electrical dipole contained in the N-terminus. We further explore the mechanism of polarity determination by utilizing the one-dimensional Poisson-Nernst-Plank model to determine the voltage profile of simple model channels containing regions of permanent charge within the channel pore. These considerations demonstrate how local variations in the electric field may influence the polarity and sensitivity of V j-gating but are unlikely to account for the appearance of bipolar V j-gating.

CO2 Sensitivity of Voltage Gating and Gating Polarity of GapJunction Channels—Connexin40 and its COOH-Terminus-Truncated Mutant

The CO(2) sensitivity of transjunctional voltage ( V(j)) gating was studied by dual voltage clamp in oocytes expressing mouse Cx40 or its COOH terminus (CT)-truncated mutant (Cx40-TR). V(j) sensitivity, determined by a standard V(j) protocol (20 mV V(j) steps, 120 mV maximal), decreased significantly with exposure to 30% CO(2). The Boltzmann values of control versus CO(2)-treated oocytes were: V(0) = 36.3 and 48.7 mV, n = 5.4 and 3.7, and G(j min) = 0.21 and 0.31, respectively. CO(2) also affected the kinetics of V(j)-dependent inactivation of junctional current ( I(j)); the time constants of two-term exponential I(j) decay, measured at V(j) = 60 mV, increased significantly with CO(2) application. Similar results were obtained with Cx40-TR, suggesting that CT does not play a role in this phenomenon. The sensitivity of Cx40 channels to 100% CO(2) was also unaffected by CT truncation. There is evidence that CO(2) decreases the V(j) sensitivity of Cx26, Cx50 and Cx37 as well, whereas it increases that of Cx45 and Cx32 channels. Since Cx40, Cx26, Cx50 and Cx37 gate at the positive side of V(j), whereas Cx45 and Cx32 gate at negative V(j), it is likely that V(j) behavior with respect to CO(2)-induced acidification varies depending on gating polarity, possibly involving the function of the postulated V(j) sensor (NH(2)-terminus).

Genetic Heterogeneity in Erythrokeratodermia Variabilis: Novel Mutations in the Connexin Gene GJB4 (Cx30.3) and Genotype-Phenotype Correlations

Erythrokeratodermia variabilis is an autosomal dominant genodermatosis characterized by persistent plaque-like or generalized hyperkeratosis and transient red patches of variable size, shape, and location. The disorder maps to a cluster of connexin genes on chromosome 1p34-p35.1 and, in a subset of families, results from mutations in the gene GJB3 encoding the gap junction protein connexin-31 (Cx31). A recent report suggested the involvement of another connexin gene (GJB4) in the etiology of erythrokeratodermia variabilis. In this study, we sequenced the coding region of GJB4 in 13 unrelated erythrokeratodermia variabilis families without detectable mutations in GJB3. Mutation analysis revealed six distinct missense mutations in five families and a sporadic case of erythrokeratodermia variabilis, all of which were not found in controls. Mutation G12D, identified in an extended Dutch family, lies in the predicted amino-terminus and may interfere with the flexibility of this domain, connexin selectivity, or gating polarity of gap junction channels. Other mutations (R22H, T85P, F137L, F189Y) were located in the transmembrane domains of Cx30.3, and are predicted to hinder regulation of voltage gating or alter the kinetics of channel closure. Affected individuals of two unrelated families harbored point mutations leading to amino acid substitution F137L, which was also reported in GJB3, yet the extent and severity of hyperkeratosis was milder compared to the corresponding mutation in GJB3. Two mutations (T85P, F137L) were associated with the occurrence of rapidly changing erythematous patches with prominent, circinate, or gyrate borders in affected children but not in adults, supporting the notion that this feature is specific to Cx30.3 defects. Nevertheless, we observed highly variable intrafamilial phenotypes, suggesting the strong influence of modifying genetic and epigenetic factors. In addition to pathogenic mutations, we identified several missense mutations and a 4 bp deletion within the GJB4 coding region, which might represent either inconsequential polymorphisms or recessive mutations. In conclusion, our results demonstrate genetic heterogeneity in erythrokeratodermia variabilis, and emphasize that intercellular communication mediated by both Cx31 and Cx30.3 is crucial for epidermal differentiation.

Opposing gates model for voltage gating of gap junction channels.

Gap junctions are intercellular channels that link the cytoplasm of neighboring cells. Because a gap junction channel is composed of two connexons docking head-to-head with each other, the channel voltage-gating profile is symmetrical for homotypic channels made of two identical connexons (hemichannels) and asymmetric for the heterotypic channels made of two different connexons (i.e., different connexin composition). In this study we have developed a gating model that allows quantitative characterization of the voltage gating of homotypic and heterotypic channels. This model differs from the present model in use by integrating, rather than separating, the contributions of the voltage gates of the two member connexons. The gating profile can now be fitted over the entire voltage range, eliminating the previous need for data splicing and fusion of two hemichannel descriptions, which is problematic when dealing with heterotypic channels. This model also provides a practical formula to render quantitative several previously qualitative concepts, including a similarity principle for matching a voltage gate to its host connexon, assignment of gating polarity to a connexon, and the effect of docking interactions between two member connexons in an intact gap junction channel.

Reversal of the Gating Polarity of Gap Junctions by Negative Charge Substitutions in the N-Terminus of Connexin 32

Intercellular channels formed by connexins (gap junctions) are sensitive to the application of transjunctional voltage ( V j), to which they gate by the separate actions of their serially arranged hemichannels (Harris, A. L., D. C. Spray, and M. V. L. Bennett. 1981. J. Gen. Physiol. 77:95–117). Single channel studies of both intercellular and conductive hemichannels have demonstrated the existence of two separate gating mechanisms, termed “ V j-gating” and “loop gating” (Trexler, E. B., M. V. L. Bennett, T. A. Bargiello, and V. K. Verselis. 1996. Proc. Natl. Acad. Sci. U.S.A. 93:5836–5841). In Cx32 hemichannels, V j-gating occurs at negative V j (Oh, S., J. B. Rubin, M. V. L. Bennett, V. K. Verselis, and T. A. Bargiello. 1999. J. Gen. Physiol. 114:339–364; Oh, S., C. K. Abrams, V. K. Verselis, and T. A. Bargiello. 2000. J. Gen. Physiol. 116:13–31). A negative charge substitution at the second amino acid position in the N-terminus reverses the polarity of V j-gating of Cx32 hemichannels (Verselis, V. K., C. S. Ginter, and T. A. Bargiello. 1994. Nature. 368:348–351; Oh et al., 2000. J. Gen. Physiol. 116:13–31). We report that placement of a negative charge at the 5th, 8th, 9th, or 10th position can reverse the polarity of Cx32 hemichannel V j-gating. We conclude that the 1st through 10th amino acid residues lie within the transjunctional electric field and within the channel pore, as in this position they could sense changes in V j and be largely insensitive to changes in absolute membrane potential ( V m). Conductive hemichannels formed by Cx32*Cx43E1 containing a negatively charged residue at either the 8th or 10th position display bi-polar V j-gating; that is, the open probability of hemichannels formed by these connexins is reduced at both positive and negative potentials and is maximal at intermediate voltages. In contrast, Cx32*Cx43E1 hemichannels with negative charges at either the 2nd or 5th positions are uni-polar, closing only at positive V j. The simplest interpretation of these data is that the Cx32 hemichannel can adopt at least two different open conformations. The 1st–5th residues are located within the electric field in all open channel conformations, while the 8th and 10th residues lie within the electric field in one conformation and outside the electric field in the other conformation.

Properties of Gap Junction Channels Formed by Cx46 Alone and in Combination with Cx50

Gap junctions formed of connexin46 (Cx46) and connexin50 (Cx50) in lens fiber cells are crucial for maintaining lens transparency. We determined the functional properties of homotypic Cx46, heterotypic Cx46/Cx50, and heteromeric Cx46/Cx50 channels in a communication-deficient neuroblastoma (N2A) cell line, using dual whole-cell recordings. N2A cultures were stably and/or transiently transfected with Cx46, Cx50, and green fluorescent protein (EGFP). The macroscopic voltage sensitivity of homotypic Cx46 conformed to the two-state model (Boltzmann parameters: G min = 0.11, V 0 = ± 48.1 mV, gating charge = 2). Cx46 single channels showed a main-state conductance of 140 ± 8 pS and multiple subconductance states ranging from ≤10 pS to 60 pS. Conservation of homotypic properties in heterotypic Cx46/Cx50 cell pairs allowed the determination of a positive relative gating polarity for the dominant gating mechanisms in Cx46 and Cx50. Observed gating properties were consistent with a second gating mechanism in Cx46 connexons. Moreover, rectification was observed in heterotypic cell pairs. Some cell pairs in cultures simultaneously transfected with Cx46 and Cx50 exhibited junctional properties not observed in other preparations, suggesting the formation of heteromeric channels. We conclude that different combinations of Cx46 and Cx50 within gap junction channels lead to unique biophysical properties.

Stoichiometry of transjunctional voltage-gating polarity reversal by a negative charge substitution in the amino terminus of a connexin32 chimera.

Gap junctions are intercellular channels formed by the serial, head to head arrangement of two hemichannels. Each hemichannel is an oligomer of six protein subunits, which in vertebrates are encoded by the connexin gene family. All intercellular channels formed by connexins are sensitive to the relative difference in the membrane potential between coupled cells, the transjunctional voltage (Vj), and gate by the separate action of their component hemichannels (Harris, A.L., D.C. Spray, and M.V. Bennett. 1981. J. Gen. Physiol. 77:95-117). We reported previously that the polarity of Vj dependence is opposite for hemichannels formed by two closely related connexins, Cx32 and Cx26, when they are paired to form intercellular channels (Verselis, V.K., C.S. Ginter, and T.A. Bargiello. 1994. Nature. 368:348-351). The opposite gating polarity is due to a difference in the charge of the second amino acid. Negative charge substitutions of the neutral asparagine residue present in wild-type Cx32 (Cx32N2E or Cx32N2D) reverse the gating polarity of Cx32 hemichannels from closure at negative Vj to closure at positive Vj. In this paper, we further examine the mechanism of polarity reversal by determining the gating polarity of a chimeric connexin, in which the first extracellular loop (E1) of Cx32 is replaced with that of Cx43 (Cx43E1). The resulting chimera, Cx32*Cx43E1, forms conductive hemichannels when expressed in single Xenopus oocytes and intercellular channels in pairs of oocytes (Pfahnl, A., X.W. Zhou, R. Werner, and G. Dahl. 1997. Pflügers Arch. 433:733-779). We demonstrate that the polarity of Vj dependence of Cx32*Cx43E1 hemichannels in intercellular pairings is the same as that of wild-type Cx32 hemichannels and is reversed by the N2E substitution. In records of single intercellular channels, Vj dependence is characterized by gating transitions between fully open and subconductance levels. Comparable transitions are observed in Cx32*Cx43E1 conductive hemichannels at negative membrane potentials and the polarity of these transitions is reversed by the N2E substitution. We conclude that the mechanism of Vj dependence of intercellular channels is conserved in conductive hemichannels and term the process Vj gating. Heteromeric conductive hemichannels comprised of Cx32*Cx43E1 and Cx32N2E*Cx43E1 subunits display bipolar Vj gating, closing to substates at both positive and negative membrane potentials. The number of bipolar hemichannels observed in cells expressing mixtures of the two connexin subunits coincides with the number of hemichannels that are expected to contain a single oppositely charged subunit. We conclude that the movement of the voltage sensor in a single connexin subunit is sufficient to initiate Vj gating. We further suggest that Vj gating results from conformational changes in individual connexin subunits rather than by a concerted change in the conformation of all six subunits.

Identification of a Novel Mutation R42P in the Gap Junction Protein β-3 Associated with Autosomal Dominant Erythrokeratoderma Variabilis

We report a missense mutation in the gap junction protein beta-3 (encoding Connexin 31), which was detected in only the affected members of a family in which the autosomal dominant skin disease erythrokeratoderma variabilis was segregating. The nucleotide change results in an arginine to proline substitution in codon 42. This residue is positioned on the first transmembrane/first extracellular domain of the gap junction protein with the mutation replacing a negatively charged residue with a nonpolar residue. This change may disrupt the conformation of the protein and voltage gating polarity leading to impaired channel function.

Opposite voltage gating polarities of two closely related connexins.

The molecular mechanisms underlying the voltage dependence of intercellular channels formed by the family of vertebrate gap junction proteins (connexins) are unknown. All vertebrate gap junctions are sensitive to the voltage difference between the cells, defined as the transjunctional voltage, Vj (refs 1, 2), and most appear to gate by the separate actions of their component hemichannels. The heterotypic Cx32/Cx26 junction displays an unpredicted rectification that was reported to represent a novel Vj dependence created by hemichannel interactions, mediated in part by the first extracellular loop E1 (ref. 9). Here we show that aspects of the rectification of Cx32/Cx26 junctions are explained by opposite gating polarities of the component hemichannels, and that the opposite gating polarity of Cx32 and Cx26 results from a charge difference in a single amino-acid residue located at the second position in the N terminus. We also show that charge substitutions at the border of the first transmembrane (M1) and E1 domains can reverse gating polarity and suppress the effects of a charge substitution at the N terminus. We conclude that the combined actions of residues at the N terminus and M1/E1 border form a charge complex that is probably an integral part of the connexin voltage sensor. A consistent correlation between charge substitution and gating polarity indicates that Cx26 and Cx32 voltage sensors are oppositely charged and that both move towards the cytoplasm upon hemichannel closure.