Lopez W et al. (2013), Insights on the mechanisms of Ca(2+) regulation...

XB-ART-49919

J Gen Physiol 2013 Jul 01;1421:23-35. doi: 10.1085/jgp.201210893.

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Insights on the mechanisms of Ca(2+) regulation of connexin26 hemichannels revealed by human pathogenic mutations (D50N/Y).

Lopez W , Gonzalez J , Liu Y , Harris AL , Contreras JE .

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Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca(2+) concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca(2+) regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca(2+) concentration, indicating that Ca(2+) facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca(2+)-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca(2+) insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca(2+), and (b) facilitating closing and stabilization of the closed state in the presence of Ca(2+). To explore the role of a negatively charged residue at position 50 in regulation by Ca(2+), this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)(-). D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES(-) restores the wild-type Ca(2+) regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca(2+) regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca(2+). Mutant cycle analysis supports a Ca(2+)-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca(2+) destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca(2+).

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???displayArticle.pmcLink??? PMC3691447
???displayArticle.link??? J Gen Physiol
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GO keywords: sensory perception of sound [+]

???displayArticle.disOnts??? autosomal dominant keratitis-ichthyosis-deafness syndrome
???displayArticle.omims??? KERATITIS-ICHTHYOSIS-DEAFNESS SYNDROME, AUTOSOMAL DOMINANT; KIDAD

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	Figure 1. Ca2+ modulates gating in hCx26 hemichannels. (A) Current traces elicited by a voltage pulse from â80 to 0 mV from oocytes expressing hCx26 hemichannels in the presence of different Ca2+ concentrations. (B) [Ca2+] doseâresponse relation determined from the peak tail current after a voltage pulse from â80 to 0 mV. The solid line represents the best fits of the data to a Hill equation (Eq. 2). (C) Deactivation time constants as a function of Ca2+ concentration. The solid line corresponds to a linear fit to the data. The data points represent mean Â± SEM of at least three independent measurements.
	Figure 2. Gating by Ca2+ is altered by D50N/Y mutations in hCx26 hemichannels. (A) Representative current traces elicited by a pulse to 0 mV from a holding potential of â80 mV for an oocyte expressing D50N or D50Y mutant hemichannels. Numbers 1, 2, and 3 correspond to current traces obtained in the presence of 10, 1.8, or 0.25 mM Ca2+, respectively. (B) Ca2+ doseâresponse curve for oocytes expressing D50N (open circles) or D50Y mutant (open diamonds) hemichannels. The solid and dotted lines represent the best fits to a Hill equation (Eq. 2) for wild-type (from Fig. 1 B) and D50N/Y mutant hemichannels, respectively. (C) Deactivation time constants of the corresponding tail currents at different Ca2+ concentrations for D50N (open circles) or D50Y mutants (fast and slow time constants are shown as two sets of diamonds). Dotted lines are the best linear fit to the D50N/Y mutant hemichannel data. The solid line corresponds to the linear fit of the average data for wild-type hemichannels (from Fig. 1 C). The data represent mean Â± SEM of at least three independent measurements.
	Figure 3. A negatively charged residue at position 50 is critical for regulation by Ca2+. (A) Hemichannel currents from oocytes expressing D50C mutant hemichannels in low and high extracellular Ca2+ to assess the effects of chemical modification with MTS reagents. Currents were elicited by a pulse to 0 mV from a holding potential of â80 mV before (black trace) or in the presence of MTSESâ (red traces) or MTSET+ (blue traces). (B) Holding currents obtained at â80 mV for oocytes expressing D50C mutant hemichannels incubated in low (0.25 mM) or high (10 mM) external Ca2+. The addition of MTSESâ increased the holding current in low extracellular Ca2+ (top trace) and decreased the holding current in the presence of high Ca2+ (bottom trace). (C) Deactivation time constants for D50C mutant hemichannels at different Ca2+ concentrations before (dotted line; from Fig. S3) and after chemical modification with MTSESâ (red triangles) or MTSET+ (blue triangles). Time constants obtained after MTSESâ modification coincide with the solid line (from Fig. 1 C) that corresponds with the linear fit for wild-type hemichannels. Conversely, the time constant obtained after MTSET+ overlaps with the linear fit for D50C mutants with no modification. The data represent mean Â± SEM of at least three independent measurements.
	Figure 4. Possible intersubunit salt bridge interaction between positions D50 and K61 in the open conformation. (A) Top (extracellular) view of the hCx26 hemichannel from the crystal structure (Protein Data Bank accession no. 2ZW3; Maeda et al., 2009). Positions D50 and K61 are highlighted in red and blue (in all subunits), respectively. (B) Enlargement showing the proximity between position D50 in one subunit and position K61 in the adjacent subunit. The average distance for the six pairs of D50âK61 residues is â¼4.4 Ã.
	Figure 5. Exchanging the positions of the negative (D50) and positive (K61) residues partially rescues the wild-type hCx26 regulation by Ca2+. (A) Current traces elicited by a pulse to 0 mV from a holding potential of â80 mV are shown for oocytes expressing D50K, K61D, or D50K/K61D mutant hemichannels in 1.8 mM Ca2+. (B) Deactivation time constants for oocytes expressing D50K/K61D mutant hemichannels (closed squares). The solid line corresponds to best fits to the average data for wild-type hemichannels (from Fig. 1 C). The dotted line is the best linear fit to the data from double mutant D50K/K61D hemichannels. (C) [Ca2+] doseâresponse relations for oocytes expressing D50K/K61D mutant hemichannels (closed squares). The solid and dotted lines correspond to the best fits to the data of a Hill equation (Eq. 1) for wild-type and double mutant D50K/K61D hemichannels. The data represent mean Â± SEM of at least three independent measurements.
	Figure 6. Mutant cycle analysis of KD indicates that D50 and K61 residues are coupled in a Ca2+-sensitive manner. (A) Scheme for mutant cycle analysis of wild-type and mutant hemichannels. (B) Graph shows the [Ca2+] doseâresponse relations for oocytes expressing D50N (open circles) and K61R (crossed squares) mutants, and D50N/K61R (crossed circles) double mutant hemichannels. The solid and dotted lines represent the best fits to the Hill equation for wild-type (from Fig. 1 B) and mutant channels, respectively. (C) Deactivation time constants for oocytes expressing wild-type (closed circles), D50N (open circles), and K61R (crossed square) mutants, and D50N/K61R (fast and slow time constants are shown as two sets of crossed circles) double mutant hemichannels. The solid line corresponds to the best fits to the average data for wild-type hemichannels using Eq. 3. The dotted line is the best fits to the data to Eq. 3 for single and double mutant hemichannels.

References [+] :

Abrams, Voltage opens unopposed gap junction hemichannels formed by a connexin 32 mutant associated with X-linked Charcot-Marie-Tooth disease. 2002, Pubmed, Xenbase