Gating properties of channels formed by colicin Ia in planar lipid bilayer membranes

Abstract

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.

Colicin N forms voltage- and pH-dependent channels in planar lipid bilayer membranes

“…On the other hand, no current increase was observed when negative and low positive membrane voltages were applied at pH values _> 5. Very similar effects have been described for a series of channel-forming colicins such as colicin A, B, El, Ia, Ib and K (Schein et al 1978;Weaver et al 1981;Bullock et al 1983;Pattus et al 1983;Nogueira and Varanda 1988). It should be emphasized at this point that such a voltage-dependent induction of channel activity is clearly different from the voltage-dependent gating process of incorporated channels (see below).…”

“…Recently a similar non-ohmic single-channel conductance has been reported for colicin B (Pressler et al 1986). Conversely, single colicin Ia channels have a rather linear current-voltage relationship (Nogueira and Varanda 1988).…”

“…Colicins of the El-class include colicin A, B, El, I a, I b and K which permeabilize the cytoplasmic membrane, thereby destroying the membrane potential Offprint requests to: E Pattus (Konisky 1982;Lazdunski et al 1988;. These colicins form well-defined voltage-gated ion channels in artificial membranes (Schein et al 1978;Weaver et al 1981;Cleveland et al 1983;Pressler et al t986;Nogueira and Varanda 1988). Channel-forming colicins have become a valuable tool for the study of the basic principles of membrane channels since they offer some advantages in experimental handling.…”

Colicin Biology

“…All colicin channels are voltage dependent, opening at positive voltage and closing at negative voltage. The voltage dependence is steep, signifying that several formal charges cross the electrical field to sense the voltage, although only a few quantitative studies have been undertaken (124,483), no doubt because of the complexity of the system: there are a multiplicity of rate constants, open states, and closed states. We presume that most of this complexity arises from the numerous partially inserted configurations of the protein, although some may be due to bound impurities, as was suggested by Cavard (89).…”

Translocation of inserted foreign epitopes by a channelforming protein

“…One group of colicins, which includes colicin Ia, forms ion-conducting channels in the inner membranes of susceptible strains (7); these colicins also form voltagedependent ion-conducting channels in planar lipid bilayers (8)(9)(10). We recently showed that for colicin Ia, channel opening and closing is associated with the translocation, across the membrane and back, of a segment of at least 68 aa (residues 474-541) (11,12).…”