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Voltage dependence of macroscopic and unitary currents of gap junction channels formed by mouse connexin50 expressed in rat neuroblastoma cells

Miduturu Srinivas *, Mauro Costa ¹, Yang Gao *, Alfredo Fort *, Glenn I. Fishman ² and David C. Spray *¹

The macroscopic and single channel gating characteristics of connexin (Cx) 50 gap junction channels between pairs of N2A neuroblastoma cells transfected with mouse Cx50 DNA were investigated using the dual whole-cell voltage clamp technique.

The macroscopic junctional current (I_j) of Cx50-transfected cells decayed exponentially with time in response to transjunctional voltage (V_j) steps (time constant () of ~4 s at a V_j of 30-40 mV and 100-200 ms at a V_j of 80-100 mV). The steady-state junctional conductance (g_j) was well described by a two-state Boltzmann equation. The half-inactivation voltage (V₀), the ratio of minimal to maximal g_j (g_min/g_max) and the equivalent gating charge were ± 37 mV, 0·21 and 4, respectively.

The conductance of single Cx50 channels measured using patch pipettes containing 130 mM CsCl was 220 ± 13·1 pS (12 cell pairs). A prominent residual or subconductance state corresponding to 43 ± 4·2 pS (10 cell pairs) was also observed at large V_j s.

The relationship between channel open probability (P_o) and V_j was well described by a Boltzmann relationship with parameters similar to those obtained for macroscopic g_j (V₀ = 34 mV, gating charge = 4·25, maximum P= 0·98). The ensemble average of single channel currents at V_j = 50 mV declined in a monoexponential manner ( = 905 ms), a value similar to the decline of the macroscopic I_j of Cx50 channels at the same voltage.

Ion substitution experiments indicated that Cx50 channels have a lower permeability to anions than to cations (transjunctional conductance of KCl vs. potassium glutamate (_j,KCl/_j,KGlut), 1·2; 6 cell pairs).

The results have important implications for understanding the role of connexins in tissues where Cx50 is a major gap junction component, including the lens.

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