An intracellular anion channel from rat brain microsomes which is also permeable to cations

Abstract

The use of theoretical and experimental approaches i l l the biophysical study of membrane channel prott.ins has provided an understanding of their transpt>rt functions. The study of C1selective channels has shown much variety in their functional characteristics. Based on functional properties such as voltage and agonist dependence, selectivity, conductance and sensitivity to blockers, C1channels fall within one of four categories: (i) background C1channels of low conductance (10-100 p S ) ; (ii) maxi Clchannels which display high conductance properties (> 200 p S ) ; (iii) ligand activated C1chann 1s which are activated by neurotransmitters and Ca5+; and (iv) double barrelled C1channels with an unusual gating mechanism [l]. C1selective channels show different permeability properties and much work has focussed on the selectivity filter region of these channel proteins [1,2,3]. The charge associated with the filter region and its size have been determined by varying the size of permeable anions and cations. The use of channel blockers in conjuction with permeability properties has helped define proposed structures of channel selectivity filter regions. This project has studied the ionic selectivity of an anion channel from rat brain microsomal membrane vesicles, by fusing channel containing membrane vesicles into a voltage clamped phospholipid planar bilayer. Using a series of monovalent chloride salts, it has been shown that the anion channel is permeable to cations as well as anions. The use of increasingly large cations has supported an unusual model for the channel selectivity filter region. Initial experiments used asymmetrical solutions o f KC1 (450mM KC1/2mM HepesIlmM Tris pH 7 . 4 cis: 150mM KC1/2mM Hepes/lmmM Ti-is pH 7 . 4 trans), where cis is the voltage-clamped side of the bilayer, to which vesicles were added, with the trans side grounded. Transmembrane currents were recorded over a range of holding pocrntials (OmV to + / 60 mV. cis trans). With KC1 Erev was t6 mV. which was a deviation from the expected Erev of +29 mV, predicted from the Nernst equation under similar ionic conditions. This shift in Erev indicated that K+ was also permeable in the channel along with Cl-. Using a modified version of the Goldman-Hodgkin-Katz equation [4], the permeability ratio of C1-:Kt was 1.5 the channel was permeable to K4, it was still predominantly C1' selective. The mechanism responsible for this dual permeability was examined by using cations of increasing size in an attempt to determine the size and charge of the selectivity filter. Choline C1 (Diameter -6 A ) , used at a 3:l gradient (cis > trans) showed choline+ was also permeable in this anion channel. The Erev for choline C1 was +10 mV, with a permeability ratio Cl-:choline+ of >2 (Figure 1.). As with KCl, the anion channel was Cl--selective. Experiments using a larger cation, Tris+ (-8 8, diameter), also showed it to be permeable in the anion channel. With a 3:l gradient (cis>trans) the Erev of +20 mV moved towards that of a purely Cl--selective channel. This indicated that the limiting size