Voltage opens unopposed gap junction hemichannels formed by a connexin 32 mutant associated with X-linked Charcot-Marie-Tooth disease.

The X-linked form of Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy that arises in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32), which is expressed by Schwann cells. We recently showed that Cx32 containing the CMTX-associated mutation, Ser-85-Cys (S85C), forms functional cell-cell channels in paired Xenopus oocytes. Here, we describe that this mutant connexin also shows increased opening of hemichannels in nonjunctional surface membrane. Open hemichannels may damage the cells through loss of ionic gradients and small metabolites and increased influx of Ca(2+), and provide a mechanism by which this and other mutant forms of Cx32 may damage cells in which they are expressed. Evidence for open hemichannels includes: (i) oocytes expressing the Cx32(S85C) mutant show greatly increased conductance at inside positive potentials, significantly larger than in oocytes expressing wild-type Cx32 (Cx32WT); and (ii) the induced currents are similar to those previously described for several other connexin hemichannels, and exhibit slowly developing increases with increasing levels of positivity and reversible reduction when intracellular pH is decreased or extracellular Ca(2+) concentration is increased. Although increased currents are seen, oocytes expressing Cx32(S85C) have lower levels of the protein in the surface and in total homogenates than do oocytes expressing Cx32WT; thus, under the conditions examined here, hemichannels in the surface membrane formed of the Cx32(S85C) mutant have a higher open probability than hemichannels formed of Cx32WT. This increase in functional hemichannels may damage Schwann cells and ultimately lead to loss of function in peripheral nerves of patients harboring this mutation.

Functional alterations in gap junction channels formed by mutant forms of connexin 32: evidence for loss of function as a pathogenic mechanism in the X-linked form of Charcot-Marie-Tooth disease.

CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32). In this communication, we describe the expression levels and biophysical parameters of seven mutant forms of Cx32 associated with CMTX, when expressed in paired Xenopus oocytes. Paired oocytes expressing the R15Q and H94Q mutants show junctional conductances not statistically different from that determined for Cx32WT, though both show a trend toward reduced levels. The S85C and G12S mutants induce reduced levels of junctional conductance. Three other mutants (R15W, H94Y and V139M) induce no conductance above baseline when expressed in paired oocytes. Analysis of the conductance voltage relations for these mutants shows that the reduced levels of conductance are entirely (H94Y and V139M) or partly (S85C and R15W) explicable by a reduced open probability of the mutant hemichannels. The R15Q and H94Q mutations also show alterations in the conductance voltage relations that would be expected to minimally (H94Q) or moderately (R15Q) reduce the available gap junction communication pathway. The reduction in G12S induced conductance cannot be explained by alterations in hemichannel open probability and are more likely due to reduced junction formation. These results demonstrate that many CMTX mutations lead to loss of function of Cx32. For these mutations, the loss of function model is likely to explain the pathogenesis of CMTX.

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;. 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.

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.

Mutations in connexin 32: the molecular and biophysical bases for the X-linked form of Charcot-Marie-Tooth disease.

The connexins are a family of homologous integral membrane proteins that form channels that provide a low resistance pathway for the transmission of electrical signals and the diffusion of small ions and non-electrolytes between coupled cells. Individuals carrying mutations in the gene encoding connexin 32 (Cx32), a gap junction protein expressed in the paranodal loops and Schmidt-Lantermann incisures of myelinating Schwann cells, develop a peripheral neuropathy - the X-linked form of Charcot-Marie-Tooth disease (CMTX). Over 160 different mutations in Cx32 associated with CMTX have been identified. Some mutations will lead to complete loss of function with no possibility of expression of functional channels. Some mutations in Cx32 lead to the abnormal accumulation of Cx32 proteins in the cytoplasm, particularly in the Golgi apparatus; CMTX may arise due to incorrect trafficking of Cx32 or to interference with trafficking of other proteins. On the other hand, many mutant forms of Cx32 can form functional channels. Some functional mutants have conductance voltage relationships that are disrupted to a degree which would lead to a substantial reduction in the available gap junction mediated communication pathway. Others have essentially normal steady-state g-V relations. In one of these cases (Ser26Leu), the only change introduced by the mutation is a reduction in the pore diameter from 7 A for the wild-type channel to less than 3 A for Ser26Leu. This reduction in pore diameter may restrict the passage of important signaling molecules. These findings suggest that in some, if not all cases of CMTX, loss of function of normal Cx32 is sufficient to cause CMTX.

The role of a conserved proline residue in mediating conformational changes associated with voltage gating of Cx32 gap junctions.

We have explored the role of a proline residue located at position 87 in the second transmembrane segment (TM2) of gap junctions in the mechanism of voltage-dependent gating of connexin32 (Cx32). Substitution of this proline (denoted Cx32P87) with residues G, A, or V affects channel function in a progressive manner consistent with the expectation that a proline kink (PK) motif exists in the second transmembrane segment (TM2) of this connexin. Mutations of the preceding threonine residue T86 to S, A, C, V, N, or L shift the conductance-voltage relation of wild-type Cx32, such that the mutated channels close at smaller transjunctional voltages. The observed shift in voltage dependence is consistent with a reduction in the open probability of the mutant hemichannels at a transjunctional voltage (Vj) of 0 mV. In both cases in which kinetics were examined, the time constants for reaching steady state were faster for T86N and T86A than for wild type at comparable voltages, suggesting that the T86 mutations cause the energetic destabilization of the open state relative to the other states of the channel protein. The structural underpinnings of the observed effects were explored with Monte Carlo simulations. The conformational space of TM2 helices was found to differ for the T86A, V, N, and L mutants, which produce a less bent helix ( approximately 20 degrees bend angle) compared to the wild type, which has a approximately 37 degrees bend angle. The greater bend angle of the wild-type helix reflects the propensity of the T86 residue to hydrogen bond with the backbone carbonyl of amino acid residue I82. The relative differences in propensity for hydrogen bonding of the mutants relative to the wild-type threonine residue in the constructs we studied (T86A, V, N, L, S, and C) correlate with the shift in the conductance-voltage relation observed for T86 mutations. The data are consistent with a structural model in which the open conformation of the Cx32 channel corresponds to a more bent TM2 helix, and the closed conformation corresponds to a less bent helix. We propose that the modulation of the hydrogen-bonding potential of the T86 residue alters the bend angle of the PK motif and mediates conformational changes between open and closed channel states.

Selenium deficiency in long-term total parenteral nutrition.

Although selenium is an essential trace element, it is often not routinely added to total parenteral nutrition (TPN) formulations. When selenium is not added, patients are at risk for selenium deficiency. This report describes such a patient. He had several operations for colon cancer, including a massive resection of the small bowel that resulted in a short bowel and a fistula. TPN was started after his last operation. After he was discharged, he had a normal, active lifestyle, except that he limited oral intake to water and an occasional soft drink. After 3 years of almost exclusive nourishment by TPN, he developed whitened nail beds. Investigation for possible trace element deficiency resulted in a finding that he had very low levels of selenium in his blood. He did not have any of the cardiac or skeletal muscle abnormalities that have been associated with selenium deficiency. After supplementation with selenium, his blood levels of selenium rose and the nail bed changes were reversed.

Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.

The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side.

A T. cruzi-secreted protein immunologically related to the complement component C9: evidence for membrane pore-forming activity at low pH.

Protozoan parasite T. cruzi invades cells within acidic vacuoles, but shortly afterward escapes into the cytosol. Exit from the phagosome is blocked by raising the pH of acidic compartments, suggesting that a previously described acid-active hemolysin secreted by T. cruzi might be involved in the membrane disruption process. Here we show that T. cruzi supernatants are cytotoxic for nucleated cells at pH 5.5 and contain a protein reactive with antibodies against reduced and alkylated human C9 (the ninth component of complement). The C9 cross-reactive protein (TC-TOX) copurified with the cytolytic activity, and the active fractions induced conductance steps characteristic of transmembrane ion channels in planar phospholipid bilayers. Immunocytochemical studies using antibodies against purified TC-TOX showed that the protein was localized to the luminal space of parasite-containing phagosomes. We postulate that TC-TOX, when secreted into the acidic environment of the phagosome, forms pores in the membrane, which contribute to its disruption.

Delta-endotoxins form cation-selective channels in planar lipid bilayers.

Delta-endotoxins CryIA(c) and CryIIIA, two members of a large family of toxic proteins from Bacillus thuringiensis, were each allowed to interact with planar lipid bilayers and were analyzed for their ability to form ion-conducting channels. Both of these toxins made clearly resolved channels in the membranes and exhibited several conductance states, which ranged from 200 pS to about 4000 pS (in 300 mM KCl). The channels formed by both toxins were highly cation-selective, but not ideally so. The permeability ratio of K+ to Cl- was about 25 for both channels. The ability of these proteins to form such channels may account for their toxic action on sensitive cells, and suggests that this family of toxins may act by a common mechanism.

Alteration of the pH-dependent ion selectivity of the colicin E1 channel by site-directed mutagenesis.

Colicin E1 is a soluble, bacteriocidal protein that forms voltage-gated channels in planar lipid bilayers. The channel-forming region of the 522-amino acid protein is near the COOH terminus, and contains a 35-amino acid hydrophobic segment which is presumed to be important in interacting with the membrane. We have used site-directed mutagenesis in the region immediately upstream from the hydrophobic segment to construct several functional colicin mutants in which a wild-type residue was replaced with a cysteine. We also replaced the only naturally occurring cysteine in the molecule, Cys-505, with alanine, so that synthetically introduced cysteines could unambiguously serve as targets for chemical modification. All of the replacements reported here (at positions 449, 459, 473, 505, and some combinations) resulted in a channel that had an ion selectivity (K+ versus Cl-) identical to wild type at low pH. At higher pH, however, one of these mutations, which replaced the negatively charged aspartate at position 473 (the upstream boundary of the hydrophobic segment), resulted in a channel that was less cation-selective than was wild type. When the introduced Cys-473 was reacted with iodoacetic acid, which inserted a COOH group close to the position of the missing aspartate COOH, wild-type ion selectivity was restored, suggesting that the greater cation selectivity of the wild-type channel was directly produced by the negative charge at Asp-473. By comparing the ion selectivity of the Cys-473 mutant channel to that of the wild type as a function of the pH on the cis and trans sides of the membrane, it was possible to locate residue 473 close to the cis side. Locating in this manner the positions in the channel of particular residues places important constraints on channel model building.

Gastric lipase: localization in the human stomach.

The aim of this study was to determine the range of activity and the location of lipase in the human stomach. The range of lipase activity in gastric mucosa of surgical specimens from the fundic area of 22 subjects was 594 to 3350 mU [mean, 1598 +/- 144 mU tri[3H]olein, (1 mU-1 nmol [3H]oleic acid released from tri[3H]olein per minute per milligram protein)]. For localization of activity, pinch biopsy specimens of gastric mucosa from 6 subjects were taken from the greater and lesser curvatures within 2 cm of the gastroesophageal junction (upper greater curvature and upper lesser curvature) and within 2 cm of the pylorus (lower greater curvature and lower lesser curvature). Lipase activity was higher in the upper greater curvature (405 +/- 92 mU) than in the upper lesser curvature (32 +/- 13 mU) and lowest in the antral area (16 +/- 9 mU in the lower lesser curvature and 10 +/- 2 mU in the lower greater curvature). The data show that in the human, lipase activity is localized primarily in the fundic area of the stomach. Comparison of the lipase activity levels in the gastric mucosa with lingual lipase activity levels in specimens of lingual serous glands indicates that in humans, gastric lipase is the main lipase active in the stomach.

Role of nonpancreatic lipolytic activity in exocrine pancreatic insufficiency.

Patients with exocrine pancreatic insufficiency may absorb greater than 50% of dietary fat despite the absence of measurable pancreatic lipase activity. Nonpancreatic lipolytic activity was measured in gastric and duodenal aspirates from 5 patients with exocrine pancreatic insufficiency secondary to alcoholism and in aspirates from 5 alcoholics without evidence of exocrine pancreatic dysfunction (controls). Samples were collected under fasting and postprandial conditions. All patients with exocrine pancreatic insufficiency had nonpancreatic lipolytic activity in gastric and duodenal aspirates. Lipolytic activity in gastric aspirates was not significantly different between the patients with exocrine pancreatic insufficiency and the controls during the fasting and postprandial periods. Pancreatic insufficiency was associated with significantly (p less than 0.05) higher nonpancreatic lipolytic activity in the duodenum under fasting conditions. No significant difference between the groups was found in postprandial nonpancreatic lipolytic activity. Nonpancreatic lipolytic activity accounted for approximately 90% of total lipolytic activity at the ligament of Treitz in patients with exocrine pancreatic insufficiency as opposed to 7% in the control subjects. These observations suggest a significant role for nonpancreatic lipolytic activity (lingual lipase and gastric lipase) in fat digestion in patients with pancreatic insufficiency secondary to chronic alcohol abuse.

Lingual lipase in cystic fibrosis. Quantitation of enzyme activity in the upper small intestine of patients with exocrine pancreatic insufficiency.

We have measured the level of lingual lipase activity in gastric and duodenal aspirates of five patients with cystic fibrosis (CF) and pancreatic insufficiency. Lingual lipase activity (measured in vitro by the hydrolysis of long-chain triglyceride, tri-[3H]olein, at pH 4.2 and expressed in nanomoles FFA released per milliliter aspirate per minute) and pH in gastric and duodenal aspirates were measured at 10-min intervals during a a 30-min basal period and at 15-min intervals during a 2-h period after the ingestion of a test meal. In gastric aspirates, lingual lipase activity decreased from basal levels of 200 +/- 34 nmol FFA released per milliliter per minute (similar to values reported previously in normal subjects (Hamosh M., H. L. Klaeveman, R. O. Wolf, and R. O. Scow, 1975, J. Clin. Invest., 55:908-913) to 79 +/- 15 nmol FFA/ml per min during the first postprandial hour and returned to basal levels during the second postprandial hour, (206 +/- 39 nmol FFA/ml per min). Duodenal aspirates, obtained during basal conditions, had lingual lipase activity similar to that in the stomach, 178 +/- 63 nmol FFA/ml per min. Enzyme activity levels were 56 +/- 14 and 113 +/- 29 during the first and second postprandial hours. Measurements of total lipase activity delivered to the ligament of Treitz showed that lingual lipase amounted to 91.22 +/- 4.06% of the total lipase activity in the upper small intestine during the 150-min study period. The basal and postprandial gastric pH levels in the five CF patients studied (3.2 +/- 0.44, 4.0 +/- 0.16, and 4.4 +/- 0.4 for basal and first and second postprandial hours, respectively) did not differ from previously reported values for normal subjects. The pH of duodenal aspirates was however significantly lower (P less than 0.001) in CF patients, both under basal conditions (5.0 +/- 0.26) and during the first and second postprandial hours (4.9 +/- 0.13 and 4.4 +/- 0.36, respectively), than in normal subjects. The low postprandial duodenal pH enables lingual lipase to act not only in the stomach but to continue the hydrolysis of dietary fat in the upper small intestine of CF patients. The data presented show that lingual lipase remains fully active in CF and accounts for greater than 90% of total lipase activity in the upper small intestine. We suggest that, because of low intestinal pH in CF, enzyme replacement therapy containing lingual lipase could improve fat absorption in CF patients to a greater extent than the pancreatic preparations now in use.