Pore-lining Region Research Articles

Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels.

Voltage-gated Ca2+ channels link changes in membrane potential to the delivery of Ca2+, a key second messenger for many cellular responses. Ca2+ channels show selectivity for Ca2+ over more plentiful ions such as Na+ or K+ by virtue of their high-affinity binding of Ca2+ within the pore. It has been suggested that this binding involves four conserved glutamate residues in equivalent positions in the putative pore-lining regions of repeats I-IV in the Ca2+ channel a1 subunit. We have carried out a systematic series of single amino-acid substitutions in each of these positions and find that all four glutamates participate in high-affinity binding of Ca2+ or Cd2+. Each glutamate carboxylate makes a distinct contribution to ion binding, with the carboxylate in repeat III having the strongest effect. Some single glutamate-to-lysine mutations completely abolish micromolar Ca2+ block, indicating that the pore does not possess any high-affinity binding site that acts independently of the four glutamate residues. The prevailing model of Ca2+ permeation must thus be modified to allow binding of two Ca2+ ions in close proximity, within the sphere of influence of the four glutamates. The functional inequality of the glutamates may be advantageous in allowing simultaneous interactions with multiple Ca2+ ions moving single-file within the pore. Competition among Ca2+ ions for individual glutamates, together with repulsive ion-ion electrostatic interaction, may help achieve rapid flux rates through the channel.

Is a beta-barrel model of the K+ channel energetically feasible?

A mutation of Shaker D431 or T449 strongly affects external tetraethylammonium (TEA) binding (1) whereas a mutation of Shaker T441 (2) or NGK2/DRK1 chimera L374 (equivalent to Shaker V443) and chimera V369 (3) affect internal TEA blocking in the potassium channel. Changes in Shaker T449 (1), F433, T441, T442 (4), and chimera L374 and V369 (3) affect ion conductance in the K+ channel. These facts suggest that SS1 ( 430-440 in Shaker) and SS2 (440-450 in Shaker) line the channel, passing through the cell membrane and back. Mutations of several residues just outside of the SS1SS2 region affect external charybdotoxin block (5). Mutations of chimera residues M379 and V368 (3), and Shaker D431 (1) have little or no effect on channel conductance. The K+ channel is comprised of four equivalent monomers (6). Using molecular mechanics, we addressed the following questions: (a) Could four pairs of SS1 and SS2 hydrogen bond together to form a ,-barrel which could span the bilayer and form the pore-lining region of the channel? (b) Is there a model consistent with the assumption that site directed mutagenesis of residues with side chains projecting into the barrel would be most likely to affect permeability properties? (c) Would there be room in the p-barrel for the inward projecting side chains? (d) Would water and K+ fit in the channel?

An SS1-SS2 beta-barrel structure for the voltage-activated potassium channel.

To examine the feasibility of a beta structure for the pore-lining region of the voltage-gated potassium channel, we have characterized a family of 12 antiparallel beta-barrels. Each is comprised of four identical pairs of beta-strands organized with approximate 4-fold symmetry about a channel axis. The C- and N-termini of the beta-strand pairs are assumed to be at the extracellular end of the channel, and each pair is connected by a hairpin turn at the intracellular end of the channel. The models differ in the residues located in the hairpin turn and in the orientation of the two strands of each pair in the barrel, i.e. whether the C-terminus of a pair is clockwise (CW) or counterclockwise (CCW) from the N-terminus when the channel is viewed from outside the cell. Following known structure precedents and potential energy predictions, the barrel is assumed to be right-twisting in all cases. All models have crowded layers of inward-projecting aromatic side-chains near the center of the channel which could regulate channel selectivity. The models with an odd number of amino acids in the hairpin turn have the advantage of predicting that F433 points into the barrel, but the disadvantage that V438 does not. Of these models, two of the models are most consistent with the external tetraethylammonium (TEA) block data, and of those, one (T439 CCW 3:5) is most consistent with the internal TEA block data.