Gating of Connexin Channels by transjunctional-voltage: Conformations and models of open and closed states.

Voltage is an important physiologic regulator of channels formed by the connexin gene family. Connexins are unique among ion channels in that both plasma membrane inserted hemichannels (undocked hemichannels) and intercellular channels (aggregates of which form gap junctions) have important physiological roles. The hemichannel is the fundamental unit of gap junction voltage-gating.

NMR and structural data for Connexin 32 and Connexin 26 N-terminal peptides.

In this article we present H and C chemical shift assignments, secondary structural propensity data and normalized temperature coefficient data for N-terminal peptides of Connexin 26 (Cx26), Cx26G12R and Cx32G12R mutants seen in syndromic deafness and Charcot Marie Tooth Disease respectively, published in "Structural Studies of N-Terminal Mutants of Connexin 26 and Connexin 32 Using 1H NMR Spectroscopy" (Y. Batir, T.A.

Structural studies of N-terminal mutants of Connexin 26 and Connexin 32 using (1)H NMR spectroscopy.

Alterations in gap junctions underlie the etiologies of syndromic deafness (KID) and Charcot-Marie Tooth disease (CMTX). Functional gap junctions are composed of connexin molecules with N-termini containing a flexible turn around G12, inserting the N-termini into the channel pore allowing voltage gating. The loss of this turn correlates with loss of Connexin 32 (Cx32) function by impaired trafficking to the cell membrane.

Voltage regulation of connexin channel conductance.

Voltage is an important parameter that regulates the conductance of both intercellular and plasma membrane channels (undocked hemichannels) formed by the 21 members of the mammalian connexin gene family. Connexin channels display two forms of voltage-dependence, rectification of ionic currents and voltage-dependent gating. Ionic rectification results either from asymmetries in the distribution of fixed charges due to heterotypic pairing of different hemichannels, or by channel block, arising from differences in the concentrations of divalent cations on opposite sides of the junctional plaque.

The carboxyl terminal residues 220-283 are not required for voltage gating of a chimeric connexin32 hemichannel.

Connexin hemichannels display two distinct forms of voltage-dependent gating, corresponding to the operation of Vj- or fast gates and loop- or slow gates. The carboxyl terminus (CT) of connexin 32 has been reported to be required for the operation of the Vj (fast) gates, but this conclusion was inferred from the loss of a fast kinetic component in macroscopic currents of CT-truncated intercellular channels elicited by transjunctional voltage. Such inferences are complicated by presence of both fast and slow gates in each hemichannel and the serial head-to-head arrangement of these gates in the intercellular channel.

Voltage-dependent gating of the Cx32*43E1 hemichannel: conformational changes at the channel entrances.

Voltage is an important parameter that regulates the open probability of both intercellular channels (gap junctions) and undocked hemichannels formed by members of the connexin gene family. All connexin channels display two distinct voltage-gating processes, termed loop- or slow-gating and V(j)- or fast-gating, which are intrinsic hemichannel properties. Previous studies have established that the loop-gate permeability barrier is formed by a large conformational change that reduces pore diameter in a region of the channel pore located at the border of the first transmembrane domain and first extracellular loop (TM1/E1), the parahelix (residues 42-51).

Functional requirement for a highly conserved charged residue at position 75 in the gap junction protein connexin 32.

Charcot Marie Tooth disease (CMT) is a group of inherited disorders characterized clinically by exclusively or predominantly peripheral nerve dysfunction. CMT1X, the most common form of X-linked CMT is caused by mutations in connexin 32 (Cx32). In this work, we used dual whole cell patch clamp recording to examine the functional effects of mutations at the Arg(75) position.

Structural studies of N-terminal mutants of connexin 32 using (1)H NMR spectroscopy.

The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with (1)H NMR that the structure of the N-terminus of functional connexin molecules contains a flexible turn around G12 (Arch. Biochem.

Molecular dynamics simulations of the Cx26 hemichannel: insights into voltage-dependent loop-gating.

Loop-gating is one of two voltage-dependent mechanisms that regulate the open probability of connexin channels. The loop-gate permeability barrier is formed by a segment of the first extracellular loop (E1) (the parahelix) and appears to be accompanied by straightening of the bend angle between E1 and the first transmembrane domain (TM1). Here, all-atom molecular dynamics simulations are used to identify and characterize interacting van der Waals and electrostatic networks that stabilize the parahelices and TM1/E1 bend angles of the open Cx26 hemichannel.

Molecular dynamics simulations of the Cx26 hemichannel: evaluation of structural models with Brownian dynamics.

The recently published crystal structure of the Cx26 gap junction channel provides a unique opportunity for elucidation of the structure of the conductive connexin pore and the molecular determinants of its ion permeation properties (conductance, current-voltage [I-V] relations, and charge selectivity). However, the crystal structure was incomplete, most notably lacking the coordinates of the N-terminal methionine residue, which resides within the pore, and also lacking two cytosolic domains. To allow computational studies for comparison with the known channel properties, we completed the structure.

Voltage-dependent conformational changes in connexin channels.

Channels formed by connexins display two distinct types of voltage-dependent gating, termed V(j)- or fast-gating and loop- or slow-gating. Recent studies, using metal bridge formation and chemical cross-linking have identified a region within the channel pore that contributes to the formation of the loop-gate permeability barrier. The conformational changes are remarkably large, reducing the channel pore diameter from 15 to 20Å to less than 4Å.

Structural studies of the N-terminus of Connexin 32 using 1H NMR spectroscopy.

The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with (1)H NMR that the structure of the N-terminus of a representative connexin molecule contains a flexible turn around glycine 12 [P.E.

Conformational changes in a pore-forming region underlie voltage-dependent "loop gating" of an unapposed connexin hemichannel.

The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA-biotin-X, when the channel resides in the open state.

Connexin 32 increases the proliferative response of Schwann cells to neuregulin-1 (Nrg1).

Connexin 32 (Cx32), a gap junction protein, is found within the para-nodal region and Schmidt-Lanterman incisures of myelinating Schwann cells (SCs). In developing and regenerating peripheral nerves, pro-myelinating SCs express Cx32 mRNA and protein in conjunction with the expression of myelin specific genes. Neuregulin-1 (Nrg1), a member of the neuregulin family of growth factors, controls SC proliferation and differentiation depending on the cellular environment and the particular stage of SC maturation.

Loop gating of connexin hemichannels involves movement of pore-lining residues in the first extracellular loop domain.

Unapposed connexin hemichannels exhibit robust closure in response to membrane hyperpolarization and extracellular calcium. This form of gating, termed "loop gating," is largely responsible for regulating hemichannel opening, thereby preventing cell damage through excessive flux of ions and metabolites. The molecular components and structural rearrangements underlying loop gating remain unknown.

Charges dispersed over the permeation pathway determine the charge selectivity and conductance of a Cx32 chimeric hemichannel.

Previous studies have shown that charge substitutions in the amino terminus of a chimeric connexin, Cx32*43E1, which forms unapposed hemichannels in Xenopus oocytes, can result in a threefold difference in unitary conductance and alter the direction and amount of open channel current rectification. Here, we determine the charge selectivity of Cx32*43E1 unapposed hemichannels containing negative and/or positive charge substitutions at the 2nd, 5th and 8th positions in the N-terminus. Unlike Cx32 intercellular channels, which are weakly anion selective, the Cx32*43E1 unapposed hemichannel is moderately cation selective.

Properties of human connexin 31, which is implicated in hereditary dermatological disease and deafness.

The connexins are a family of at least 20 homologous proteins in humans that form aqueous channels connecting the interiors of coupled cells and mediating electrical and chemical communication. Mutations in the gene for human connexin 31 (hCx31) are associated with disorders of the skin and auditory system. Alterations in functional properties of Cx31 junctions are likely to play a role in these diseases; nonetheless, little is known about the properties of the wild-type channels.

Correlative studies of gating in Cx46 and Cx50 hemichannels and gap junction channels.

Transjunctional voltage (V(j)) gating of gap junction (GJ) channels formed of connexins has been proposed to occur by gating of the component hemichannels. We took advantage of the ability of Cx46 and Cx50 to function as unapposed hemichannels to identify gating properties intrinsic to hemichannels and how they contribute to gating of GJ channels. We show that Cx46 and Cx50 hemichannels contain two distinct gating mechanisms that generate reductions in conductance for both membrane polarities.

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.

Pore-lining residues identified by single channel SCAM studies in Cx46 hemichannels.

The substituted cysteine accessibility method was applied to single Cx46 hemichannels to identify residues that participate in lining the aqueous pore of channels formed of connexins. Criteria for assignment to the pore included reactivity to sulfydryl-specific methanethiosulfonate (MTS) reagents from both sides of an open hemichannel and observable effects on open channel properties. We demonstrate reactivity to MTS reagents over a stretch of seventeen amino acids, D51 through L35, that constitute segments of E1 and TM1.

Pathogenesis of X-linked Charcot-Marie-Tooth disease: differential effects of two mutations in connexin 32.

X-linked Charcot-Marie-Tooth disease is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding connexin 32 (Cx32). Cx32 is expressed at the paranodes and Schmidt-Lantermann incisures of myelinating Schwann cells in which it is believed to form a reflexive pathway between the abaxonal and adaxonal cytoplasmic domains. Patients with the Val181Ala (V181A) mutation have a severe peripheral neuropathy.

Single-channel SCAM identifies pore-lining residues in the first extracellular loop and first transmembrane domains of Cx46 hemichannels.

Gap junction (GJ) channels provide an important pathway for direct intercellular transmission of signaling molecules. Previously we showed that fixed negative charges in the first extracellular loop domain (E1) strongly influence charge selectivity, conductance, and rectification of channels and hemichannels formed of Cx46. Here, using excised patches containing Cx46 hemichannels, we applied the substituted cysteine accessibility method (SCAM) at the single channel level to residues in E1 to determine if they are pore-lining.

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.

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 first extracellular loop domain is a major determinant of charge selectivity in connexin46 channels.

Intercellular channels formed of members of the gene family of connexins (Cxs) vary from being substantially cation selective to being anion selective. We took advantage of the ability of Cx46 to function as an unopposed hemichannel to examine the basis of Cx charge selectivity. Previously we showed Cx46 hemichannels to be large pores that predominantly conduct cations and inwardly rectify in symmetric salts, properties suggesting selectivity is influenced by fixed negative charges located toward the extracellular end of the pore.