Calmodulin-Cork Model of Gap Junction Channel Gating—One Molecule, Two Mechanisms

The Calmodulin-Cork gating model is based on evidence for the direct role of calmodulin (CaM) in channel gating. Indeed, chemical gating of cell-to-cell channels is sensitive to nanomolar cytosolic calcium concentrations [Ca2+]i. Calmodulin inhibitors and inhibition of CaM expression prevent chemical gating. CaMCC, a CaM mutant with higher Ca2+-sensitivity greatly increases chemical gating sensitivity (in CaMCC the NH2-terminal EF-hand pair (res. 9–76) is replaced by the COOH-terminal pair (res. 82–148). Calmodulin colocalizes with connexins. Connexins have high-affinity CaM binding sites. Several connexin mutants paired to wild-type connexins have a high gating sensitivity that is eliminated by inhibition of CaM expression. Repeated transjunctional voltage (Vj) pulses slowly and progressively close a large number of channels by the chemical/slow gate (CaM lobe). At the single-channel level, the chemical/slow gate closes and opens slowly with on-off fluctuations. The model proposes two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, gating involves Ca2+-induced CaM-activation. In the second, gating takes place without [Ca2+]i rise. The Ca-CaM-Cork gating is only reversed by a return of [Ca2+]i to resting values, while the CaM-Cork gating is reversed by Vj positive at the gated side.

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Calmodulin-Connexin Partnership in Gap Junction Channel Regulation-Calmodulin-Cork Gating Model

International Journal of Molecular Sciences ◽

10.3390/ijms222313055 ◽

2021 ◽

Vol 22 (23) ◽

pp. 13055

Author(s):

Camillo Peracchia ◽

Lillian Mae Leverone Peracchia

Keyword(s):

Gap Junction ◽

Single Channel ◽

Wild Type ◽

X Ray Diffraction ◽

The Past ◽

Channel Regulation ◽

Gating Model ◽

Gap Junction Channel ◽

Chemical Gating ◽

Nanomolar Range

In the past four decades numerous findings have indicated that gap junction channel gating is mediated by intracellular calcium concentrations ([Ca2+i]) in the high nanomolar range via calmodulin (CaM). We have proposed a CaM-based gating model based on evidence for a direct CaM role in gating. This model is based on the following: CaM inhibitors and the inhibition of CaM expression to prevent chemical gating. A CaM mutant with higher Ca2+ sensitivity greatly increases gating sensitivity. CaM co-localizes with connexins. Connexins have high-affinity CaM-binding sites. Connexin mutants paired to wild type connexins have a higher gating sensitivity, which is eliminated by the inhibition of CaM expression. Repeated trans-junctional voltage (Vj) pulses progressively close channels by the chemical/slow gate (CaM’s N-lobe). At the single channel level, the gate closes and opens slowly with on-off fluctuations. Internally perfused crayfish axons lose gating competency but recover it by the addition of Ca-CaM to the internal perfusion solution. X-ray diffraction data demonstrate that isolated gap junctions are gated at the cytoplasmic end by a particle of the size of a CaM lobe. We have proposed two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, the gating involves Ca2+-induced CaM activation. In the second, the gating occurs without a [Ca2+]i rise.

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Calmodulin-connexin colocolization and direct role of calmodulin in gap junction channel gating

Neurophysiology ◽

10.1007/bf02506538 ◽

2000 ◽

Vol 32 (3) ◽

pp. 132-133

Author(s):

A. Sotkis ◽

X. G. Wang ◽

L. L. Peracchia ◽

A. J. Persechini ◽

C. Peracchia

Keyword(s):

Gap Junction ◽

Channel Gating ◽

Direct Role ◽

Gap Junction Channel

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Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

The Journal of General Physiology ◽

10.1085/jgp.200810056 ◽

2008 ◽

Vol 132 (6) ◽

pp. 681-692 ◽

Cited By ~ 54

Author(s):

Qinghuan Xiao ◽

Andrew Prussia ◽

Kuai Yu ◽

Yuan-yuan Cui ◽

H. Criss Hartzell

Keyword(s):

Amino Acids ◽

Density Functional ◽

Transmembrane Domain ◽

Channel Gating ◽

Regulatory Domain ◽

Acidic Amino Acids ◽

C Terminus ◽

Ef Hand ◽

Cl Channels

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl− ion channels that are activated by intracellular Ca2+. At present, the structures and mechanisms responsible for Ca2+ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca2+ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca2+ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca2+ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca2+. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca2+ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca2+-binding loop (312–323) reduced the apparent Ca2+ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca2+-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca2+ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca2+ and suggest a model in which Ca2+ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ∼100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca2+ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca2+.

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Role of Amino-terminal Half of the S4-S5 Linker in Type 1 Ryanodine Receptor (RyR1) Channel Gating

Journal of Biological Chemistry ◽

10.1074/jbc.m111.255240 ◽

2011 ◽

Vol 286 (41) ◽

pp. 35571-35577 ◽

Cited By ~ 14

Author(s):

Takashi Murayama ◽

Nagomi Kurebayashi ◽

Toshiharu Oba ◽

Hideto Oyamada ◽

Katsuji Oguchi ◽

...

Keyword(s):

Ryanodine Receptor ◽

Single Channel ◽

Signal Transmission ◽

Channel Gating ◽

Helical Structure ◽

Release Channel ◽

Amino Terminal ◽

Molecular Events

The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca2+ itself, i.e. Ca2+-induced Ca2+ release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr4825–Ser4829) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca2+ release, single-channel current recordings, and [3H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca2+ sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.

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Gap Junction Channelopathies and Calmodulinopathies. Do Disease-Causing Calmodulin Mutants Affect Direct Cell–Cell Communication?

International Journal of Molecular Sciences ◽

10.3390/ijms22179169 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9169

Author(s):

Camillo Peracchia

Keyword(s):

Gap Junction ◽

Genetic Diseases ◽

Channel Gating ◽

Cell Communication ◽

Potential Effect ◽

Gap Junction Channel ◽

Direct Cell ◽

Connexin Genes ◽

Cell Cell

The cloning of connexins cDNA opened the way to the field of gap junction channelopathies. Thus far, at least 35 genetic diseases, resulting from mutations of 11 different connexin genes, are known to cause numerous structural and functional defects in the central and peripheral nervous system as well as in the heart, skin, eyes, teeth, ears, bone, hair, nails and lymphatic system. While all of these diseases are due to connexin mutations, minimal attention has been paid to the potential diseases of cell–cell communication caused by mutations of Cx-associated molecules. An important Cx accessory protein is calmodulin (CaM), which is the major regulator of gap junction channel gating and a molecule relevant to gap junction formation. Recently, diseases caused by CaM mutations (calmodulinopathies) have been identified, but thus far calmodulinopathy studies have not considered the potential effect of CaM mutations on gap junction function. The major goal of this review is to raise awareness on the likely role of CaM mutations in defects of gap junction mediated cell communication. Our studies have demonstrated that certain CaM mutants affect gap junction channel gating or expression, so it would not be surprising to learn that CaM mutations known to cause diseases also affect cell communication mediated by gap junction channels.

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Chimeric evidence for a role of the connexin cytoplasmic loop in gap junction channel gating

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s004240050076 ◽

1996 ◽

Vol 431 (6) ◽

pp. 844-852 ◽

Cited By ~ 41

Author(s):

Xiaoguang Wang ◽

Liqiong Li ◽

Lillian L. Peracchia ◽

Camillo Peracchia

Keyword(s):

Gap Junction ◽

Channel Gating ◽

Cytoplasmic Loop ◽

Gap Junction Channel

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NMDA receptor channel gating control by the pre-M1 helix

The Journal of General Physiology ◽

10.1085/jgp.201912362 ◽

2020 ◽

Vol 152 (4) ◽

Cited By ~ 8

Author(s):

Miranda J. McDaniel ◽

Kevin K. Ogden ◽

Steven A. Kell ◽

Pieter B. Burger ◽

Dennis C. Liotta ◽

...

Keyword(s):

Nmda Receptor ◽

Conformational Changes ◽

Time Course ◽

Transmembrane Domain ◽

Single Channel ◽

De Novo ◽

Channel Gating ◽

Open Probability ◽

Single Channel Currents

The NMDA receptor (NMDAR) is an ionotropic glutamate receptor formed from the tetrameric assembly of GluN1 and GluN2 subunits. Within the flexible linker between the agonist binding domain (ABD) and the M1 helix of the pore-forming transmembrane helical bundle lies a two-turn, extracellular pre-M1 helix positioned parallel to the plasma membrane and in van der Waals contact with the M3 helix thought to constitute the channel gate. The pre-M1 helix is tethered to the bilobed ABD, where agonist-induced conformational changes initiate activation. Additionally, it is a locus for de novo mutations associated with neurological disorders, is near other disease-associated de novo sites within the transmembrane domain, and is a structural determinant of subunit-selective modulators. To investigate the role of the pre-M1 helix in channel gating, we performed scanning mutagenesis across the GluN2A pre-M1 helix and recorded whole-cell macroscopic and single channel currents from HEK293 cell-attached patches. We identified two residues at which mutations perturb channel open probability, the mean open time, and the glutamate deactivation time course. We identified a subunit-specific network of aromatic amino acids located in and around the GluN2A pre-M1 helix to be important for gating. Based on these results, we are able to hypothesize about the role of the pre-M1 helix in other NMDAR subunits based on sequence and structure homology. Our results emphasize the role of the pre-M1 helix in channel gating, implicate the surrounding amino acid environment in this mechanism, and suggest unique subunit-specific contributions of pre-M1 helices to GluN1 and GluN2 gating.

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Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons.

The Journal of General Physiology ◽

10.1085/jgp.105.6.837 ◽

1995 ◽

Vol 105 (6) ◽

pp. 837-859 ◽

Cited By ~ 5

Author(s):

J L Donnelly ◽

B S Pallotta

Keyword(s):

Steady State ◽

Rat Brain ◽

Cortical Neurons ◽

Single Channel ◽

Channel Gating ◽

Reversal Potential ◽

Open Probability ◽

Histidine Residues ◽

Gating Model ◽

Single Channel Currents

The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n-acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more extracellular histidine residues involved in channel gating are associated with a single kinetic state.

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