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Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells.

Valiunas V - J. Gen. Physiol. (2002)

Bottom Line: These currents did not occur in non-transfected cells.No significant dye uptake was detected in non-transfected cells.The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca(2+) concentration is reduced.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794, USA. virgis@mail.pnb.sunysb.edu

ABSTRACT
Human HeLa cells transfected with mouse Cx45 and rat RIN cells transfected with chicken Cx45 were used to study the electrical and permeability properties of Cx45 gap junction hemichannels. With no extracellular Ca(2+), whole-cell recording revealed currents arising from hemichannels in both transfected cell lines. Multichannel currents showed a time-dependent activation or deactivation sensitive to voltage, V(m). These currents did not occur in non-transfected cells. The hemichannel currents were inhibited by raising extracellular Ca(2+) or by acidification with CO(2). The unitary conductance exhibited V(m) dependence (i.e., gamma(hc,main) increased/decreased with hyperpolarization/depolarization). Extrapolation to V(m) = 0 mV led to a gamma(hc,main) of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, P(o), was V(m)-dependent, declining at negative V(m) (P(o) < 0.11, V(m) < -50 mV), and increasing at positive V(m) (P(o) approximately 0.76, V(m) > 50 mV). Moreover, Cx45 nonjunctional hemichannels appeared to mediate lucifer yellow (LY) and propidium iodide (PI) dye uptake from the external solution when extracellular Ca(2+) level was reduced. Dye uptake was directly proportional to the number of functioning hemichannels. No significant dye uptake was detected in non-transfected cells. Cx45 transfected HeLa and RIN cells also allowed dye to leak out when preloaded with LY and then incubated in Ca(2+)-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca(2+). Intact Cx45 gap junction channels allowed passage of either LY or PI dye, but their respective flux rates were different. Comparison of LY diffusion through Cx45 hemichannels and intact gap junction channels revealed that the former is more permeable, suggesting that gap junction channel pores exhibit more allosterical restriction to the dye molecules than the unopposed hemichannel. The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca(2+) concentration is reduced.

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Multi versus single hemichannel currents, Ihc, recorded from Cx45-HeLa cell with no Ca2+ added to the bath. (A) Multichannel Ihc, elicited by symmetrical biphasic 50 mV pulses from a holding potential of 0 mV. (B) Single-channel Ihc (bottom trace), elicited by depolarization from 0 to 30 mV (top trace). Ihc shows discrete steps indicative of opening and closing of hemichannels. The current steps yielded a conductance of ∼49–51 pS. (C) Single-channel Ihc (bottom trace), induced by hyperpolarization from +50 to −50 mV (top trace). Ihc showed discrete steps indicative of sequential closure of hemichannels. The current histograms yielded a conductance of 65–73 pS. (D–F) Hemichannel currents, Ihc, from a Cx45-HeLa cell, recorded in the cell-attached patch configuration. (D) V = ± 70 mV; (E) V = ± 90 mV; (F) V = −70 mV. The current histograms represent the following conductive states: o, hemichannel open; c, hemichannel closed; and I = 0, zero current level. Based on o-c currents, the current histograms yielded the following conductances: 52/63 pS (positive/negative V) in D and 50/66 pS (positive/negative V) in E. (F) Ihc exhibits two prominent levels corresponding to Ihc,main (bottom dotted line) and Ihc,residual (middle dotted line). The analysis yielded the following conductances: γhc,main = 62 pS, γhc,residual = 15 pS.
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fig3: Multi versus single hemichannel currents, Ihc, recorded from Cx45-HeLa cell with no Ca2+ added to the bath. (A) Multichannel Ihc, elicited by symmetrical biphasic 50 mV pulses from a holding potential of 0 mV. (B) Single-channel Ihc (bottom trace), elicited by depolarization from 0 to 30 mV (top trace). Ihc shows discrete steps indicative of opening and closing of hemichannels. The current steps yielded a conductance of ∼49–51 pS. (C) Single-channel Ihc (bottom trace), induced by hyperpolarization from +50 to −50 mV (top trace). Ihc showed discrete steps indicative of sequential closure of hemichannels. The current histograms yielded a conductance of 65–73 pS. (D–F) Hemichannel currents, Ihc, from a Cx45-HeLa cell, recorded in the cell-attached patch configuration. (D) V = ± 70 mV; (E) V = ± 90 mV; (F) V = −70 mV. The current histograms represent the following conductive states: o, hemichannel open; c, hemichannel closed; and I = 0, zero current level. Based on o-c currents, the current histograms yielded the following conductances: 52/63 pS (positive/negative V) in D and 50/66 pS (positive/negative V) in E. (F) Ihc exhibits two prominent levels corresponding to Ihc,main (bottom dotted line) and Ihc,residual (middle dotted line). The analysis yielded the following conductances: γhc,main = 62 pS, γhc,residual = 15 pS.

Mentions: Transfected HeLa cells and RIN cells often showed Ihc signals from tens of hemichannels. They were used to examine multichannel currents. Fig. 3 compares multichannel and single-channel currents through hemichannels. The cell was superfused with Ca2+-free solution. Fig. 3 A shows multichannel currents elicited by biphasic pulse (±50 mV; top trace). Depolarization gave rise to a time-dependent increase in Ihc exhibiting discrete current steps. About 9–10 hemichannels were involved during this episode. Hyperpolarization led to a large Ihc,inst which decreased rapidly to a level attributable to endogenous channels. With this time resolution no discrete current steps could be resolved.


Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells.

Valiunas V - J. Gen. Physiol. (2002)

Multi versus single hemichannel currents, Ihc, recorded from Cx45-HeLa cell with no Ca2+ added to the bath. (A) Multichannel Ihc, elicited by symmetrical biphasic 50 mV pulses from a holding potential of 0 mV. (B) Single-channel Ihc (bottom trace), elicited by depolarization from 0 to 30 mV (top trace). Ihc shows discrete steps indicative of opening and closing of hemichannels. The current steps yielded a conductance of ∼49–51 pS. (C) Single-channel Ihc (bottom trace), induced by hyperpolarization from +50 to −50 mV (top trace). Ihc showed discrete steps indicative of sequential closure of hemichannels. The current histograms yielded a conductance of 65–73 pS. (D–F) Hemichannel currents, Ihc, from a Cx45-HeLa cell, recorded in the cell-attached patch configuration. (D) V = ± 70 mV; (E) V = ± 90 mV; (F) V = −70 mV. The current histograms represent the following conductive states: o, hemichannel open; c, hemichannel closed; and I = 0, zero current level. Based on o-c currents, the current histograms yielded the following conductances: 52/63 pS (positive/negative V) in D and 50/66 pS (positive/negative V) in E. (F) Ihc exhibits two prominent levels corresponding to Ihc,main (bottom dotted line) and Ihc,residual (middle dotted line). The analysis yielded the following conductances: γhc,main = 62 pS, γhc,residual = 15 pS.
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Related In: Results  -  Collection

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fig3: Multi versus single hemichannel currents, Ihc, recorded from Cx45-HeLa cell with no Ca2+ added to the bath. (A) Multichannel Ihc, elicited by symmetrical biphasic 50 mV pulses from a holding potential of 0 mV. (B) Single-channel Ihc (bottom trace), elicited by depolarization from 0 to 30 mV (top trace). Ihc shows discrete steps indicative of opening and closing of hemichannels. The current steps yielded a conductance of ∼49–51 pS. (C) Single-channel Ihc (bottom trace), induced by hyperpolarization from +50 to −50 mV (top trace). Ihc showed discrete steps indicative of sequential closure of hemichannels. The current histograms yielded a conductance of 65–73 pS. (D–F) Hemichannel currents, Ihc, from a Cx45-HeLa cell, recorded in the cell-attached patch configuration. (D) V = ± 70 mV; (E) V = ± 90 mV; (F) V = −70 mV. The current histograms represent the following conductive states: o, hemichannel open; c, hemichannel closed; and I = 0, zero current level. Based on o-c currents, the current histograms yielded the following conductances: 52/63 pS (positive/negative V) in D and 50/66 pS (positive/negative V) in E. (F) Ihc exhibits two prominent levels corresponding to Ihc,main (bottom dotted line) and Ihc,residual (middle dotted line). The analysis yielded the following conductances: γhc,main = 62 pS, γhc,residual = 15 pS.
Mentions: Transfected HeLa cells and RIN cells often showed Ihc signals from tens of hemichannels. They were used to examine multichannel currents. Fig. 3 compares multichannel and single-channel currents through hemichannels. The cell was superfused with Ca2+-free solution. Fig. 3 A shows multichannel currents elicited by biphasic pulse (±50 mV; top trace). Depolarization gave rise to a time-dependent increase in Ihc exhibiting discrete current steps. About 9–10 hemichannels were involved during this episode. Hyperpolarization led to a large Ihc,inst which decreased rapidly to a level attributable to endogenous channels. With this time resolution no discrete current steps could be resolved.

Bottom Line: These currents did not occur in non-transfected cells.No significant dye uptake was detected in non-transfected cells.The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca(2+) concentration is reduced.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794, USA. virgis@mail.pnb.sunysb.edu

ABSTRACT
Human HeLa cells transfected with mouse Cx45 and rat RIN cells transfected with chicken Cx45 were used to study the electrical and permeability properties of Cx45 gap junction hemichannels. With no extracellular Ca(2+), whole-cell recording revealed currents arising from hemichannels in both transfected cell lines. Multichannel currents showed a time-dependent activation or deactivation sensitive to voltage, V(m). These currents did not occur in non-transfected cells. The hemichannel currents were inhibited by raising extracellular Ca(2+) or by acidification with CO(2). The unitary conductance exhibited V(m) dependence (i.e., gamma(hc,main) increased/decreased with hyperpolarization/depolarization). Extrapolation to V(m) = 0 mV led to a gamma(hc,main) of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, P(o), was V(m)-dependent, declining at negative V(m) (P(o) < 0.11, V(m) < -50 mV), and increasing at positive V(m) (P(o) approximately 0.76, V(m) > 50 mV). Moreover, Cx45 nonjunctional hemichannels appeared to mediate lucifer yellow (LY) and propidium iodide (PI) dye uptake from the external solution when extracellular Ca(2+) level was reduced. Dye uptake was directly proportional to the number of functioning hemichannels. No significant dye uptake was detected in non-transfected cells. Cx45 transfected HeLa and RIN cells also allowed dye to leak out when preloaded with LY and then incubated in Ca(2+)-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca(2+). Intact Cx45 gap junction channels allowed passage of either LY or PI dye, but their respective flux rates were different. Comparison of LY diffusion through Cx45 hemichannels and intact gap junction channels revealed that the former is more permeable, suggesting that gap junction channel pores exhibit more allosterical restriction to the dye molecules than the unopposed hemichannel. The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca(2+) concentration is reduced.

Show MeSH
Related in: MedlinePlus