Colicin Ia forms voltage-dependent channels when incorporated into planar lipid bilayers. A membrane containing many Colicin Ia channels shows a conductance which is turned on when high positive voltages (greater than +10 mV) are applied to the cis side (side to which the protein is added). The ionic current flowing through the membrane in response to a voltage step shows at first an exponential and then a linear rise with time. The relationship between the steady-state conductance, achieved immediately after the exponential portion, and voltage is S-shaped and is adequately fit by a Boltzmann distribution. The time constant (tau) of the exponential is also dependent on voltage, and the relation between these two parameters is asymmetric around Vo (voltage at which half of the channels are open). In both cases the steepness of the voltage dependence, a consequence of the number of effective gating particles (n) present in the channel, is greatly influenced by the pH of the bathing solutions. Thus, increasing the pH leads to a reduction in n, while acidic pH's have the opposite effects. This result is obtained either by changing the pH on both sides of the membrane or on only one side, be it cis or trans. On the other hand, changing pH on only one side by addition of an impermeant buffer fails to induce any change in n. At the single-channel level, pH had an effect both on the unitary conductance, doubling it in going from pH 4.5 to 8.2, as well as on the fraction of time the channels stay open, F(v). For a given voltage, F(v) is clearly diminished by increasing the pH. This titration of the voltage sensitivity leads to the conclusion that gating in the Colicin Ia molecule is accomplished by charged amino- acid residues present in the protein molecule. Our results also support the notion that these charged groups are inside the aqueous portion of the channel.

When the Na electrochemical potential difference across the skin (delta muNa) is altered by perturbing the transmembrane electrical potential difference or the external Na concentration, effects on transport and associated oxygen consumption can be described by the formalism of linear nonequilibrium thermodynamics (Vieira, Caplan & Essig, 1972, J. Gen. Physiol. 59:77; Danisi & Lacaz-Vieira, 1974, J. Gen. Physiol. 64:372; Procópio and Lacaz-Vieira, 1977, J. Membrane Biol. 35:219). We now show that with modifications of delta muNa by substitution of Li or choline for Na in the inner bathing solution, this formalism is no longer applicable. Inner Na by K substitution ((Na X K)i) causes profound alterations in short-circuit current (SCC), JinNa, K efflux (JeffK) and PD. SCC drops transiently after (Na X K)i in Cl and in SO4 media, increasing subsequently. In Cl medium, following the initial transient, there is a late decline in SCC toward a steady state. The rate of SCC decline in Cl medium is more pronounced than that observed in SO4 medium. (Na X K)i causes a transient increase in JinNa with a peak synchronous to the minimum in SCC, both in Cl and in SO4 media. This was interpreted as due to depolarization of the inner membrane. In SO4 medium, following the peak observed after (Na X K)i, JimNa drops, to increase again toward a steady state in which SCC and JinNa are not statistically different, resembling the control condition before (Na X K)i. In Cl medium, however, the JinNa steady state is approximately 100% higher than SCC. This difference is due to an important K efflux (JeffK), which builds up progressively after the substitution. The apparent K permeability [JeffK/(Ki)] is of comparable magnitude in Cl and in SO4 media before (Na X K)i and also in SO4 medium after (Na X K)i. However, in Cl medium, after (Na X K)i, the apparent K permeability increases one order of magnitude as compared to the control condition before the ionic substitution. In Cl medium, the high levels of JinNa and of Jeff(K) observed in the steady state after (Na X K)i were interpreted as being a consequence of cell swelling. SCC and PD follow very different temporal patterns after (Na X K)i which are characterized by transients in SCC and a simple fall in PD. Reasons for these differences are discussed.