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L. Ebihara, J.–J. Tong; The N–Terminus Is an Important Determinant of Both Hemichannel and Gap Junctional Channel Properties . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1856.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Gap junctional channels and hemichannels formed from the lens fiber gap junctional proteins, Cx46 and Cx50 (or its chicken orthologues, Cx56 and Cx45.6), exhibit marked differences in gating and unitary channel conductance. We have recently shown that the N–terminus is largely responsible for the differences in gap junctional channel properties. The purpose of this study is to determine the effect of exchanging the N–terminus or the first transmembrane domain(M1) on hemichannel properties and to correlate the properties of the hemichannels with those of the gap junctional channels. Methods: A series of constructs including Cx45.6 and chimeras in which the N–terminus of Cx45.6 was replaced by the corresponding region of Cx56 (Cx45.6*56N) or the first transmembrane spanning domain of Cx45.6 was replaced by the corresponding domain of Cx56 (Cx45.6*56M1) were expressed in Xenopus oocytes. Hemichannel currents were recorded in single oocytes using the two–microelectrode–voltage–clamp–technique. Results: Oocytes expressing Cx45.6 or Cx45.6*56N exhibit hemichannel currents that activate when the external calcium concentration is reduced below .2 mM. In recording solutions containing zero added calcium and 1 mM Mg+2, both types of channels have a maximum value of Po near 0 mV and tend to close on application of large positive or negative potentials. However, Cx45.6 hemichannels have a very different voltage dependence from Cx45.6*56N. At negative potentials, Cx45.6 hemichannel currents close much more slowly and less completely than Cx45.6*56N whereas at large positive potentials, Cx45.6 currents close more rapidly and to a lower steady–state level than Cx45.6*56N. In contrast, exchanging M1 results in hemichannel currents whose voltage dependence and gating kinetics are indistinguishable those of wild–type Cx45.6 at negative potentials but whose size is markedly increased. This increase in size could be at least in part attributed to a decrease in the sensitivity of Cx45.6*56M1 currents to block by external calcium. Changing [Ca]o does not alter the gating kinetics of the Cx45.6*56M1 currents. Comparison of the voltage–dependent gating properties of mutant and wild–type hemichannels with the Vj–gating properties of their corresponding gap junctional channels demonstrate that the kinetics of channel closure and the steady–state I–V curve of the hemichannels more closely resemble those of their respective gap junctional channels at positive potentials. Conclusions: The N–terminus is an important determinant of gap junctional channel and hemichannel gating.
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