The Channel Gate

The Streptomyces lividans K+ channel (KcsA) is a 160-residue protein that forms homotetrameric channels closely related to the pore domain of larger voltage-dependent channels (Schrempf et al., 1995). When purified and reconstituted in lipid bilayers, KcsA catalyzes single-channel activities with selectivity properties identical to those of other eukaryotic K+ channels (Cuello et al., 1998 Heginbotham et al., 1999 Meuser et al., 1999). The fact that KcsA is easily expressed in Escherichia coli at milligram levels made this protein an ideal target for structural analysis. 

Crystallographic studies led to the high-resolution structure of KcsA by Doyle et al. (1998b), an achievement that lent a firm structural foundation to more than three decades of functional work on K+ channels. The crystal structure revealed that KcsA is formed by the association of four subunits, contributing equally to form a water-filled pore. Each subunit has two transmembrane segments, TM1 in the periphery of the complex and TM2 lining the permeation path. Toward the extracellular face of the channel is the selectivity filter, where intimate contact with the permeant ions takes place. 

Site-Directed, Spin Label Analysis of KcsA 

Collision of nitroxides with fast-relaxing radicals, such as oxygen and metal ion complexes, causes spin exchange that effectively shortens the spin-lattice relaxation time T of the nitroxide (Hyde and Subczynski, 1989). This effect can be measured either by continuous wave (CW) power saturation techniques or by saturation recovery methods. Collision frequency is directly proportional to the accessibility of the paramagnetic reagent to the nitroxide radical and is defined as 

Two nitroxides, separated by the distance r, are coupled through space via electron-electron dipolar interactions arising from the unpaired electrons. Spin coupling induces line splitting dependent on their separation and their orientation with respect to the magnetic field according to 

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The second proton transfer mechanism involves protonation of carboxyl or histidyl groups associated with electron carriers in the membrane and release of protons from these sites through proposed channels when the electron carrier is oxidized. This is essentially a proton channel system with movement through the channel gated by the oxidation-reduction state of the prosthetic group on the electron transport protein. The classical example of this is seen in cytochrome c oxidase (Figure 3). 

Table VI lists Aa and Aa, which are estimated from Fig. 14 [y = — 1 /2, as in Eq. (62)]. We first notice that Aa of PBP is considerably larger than those of the other isomers. Equation (59) suggests one of the possible reasons An average value of (Det.V"(Q- j) may be particularly small for PBP. The geometrical meaning of this quantity has been described above. According to it, one may say that the channel gates from the PBP basin through which to pass to the neighboring basins are wide open. Another reason can be that the PBP basin may be associated with more channels connected to the other basins [see Ma in Eq. (59)]. Although the high symmetry of the PBP structure seems to make this situation possible, we have no clear evidence to support the above possibilities at this moment.

No drastic change occurred in tail sodium current. When tetramethrin was added to the BTX-treated axon, a large and prolonged tail current characteristic of the tetramethrin modified sodium channel developed. Thus tetramethrin binds to a site different from the binding site of BTX which is located inside of the channel. This result is compatible with the hypothesis that the pyrethroid molecules bind to the channel gating machinery via the membrane lipid phase thereby altering the kinetics of channel gating. 

Recent discoveries of potent non-competitive antagonists of glutamate receptors present on excitable cells of vertebrates and invertebrates offer opportunities to the chemical industry for rational development of novel therapeutics and pesticides The interactions of a number of non-competitive antagonists of locust muscle glutamate receptors with the channels gated by these membrane proteins are described and discussed qualitatively and quantitatively in terms of channel blocking kinetics. 

And finally, it is important to consider that ion channels are surrounded and directly associated with membrane phospholipids that influence the channel gating. However, the exact mechanisms of channelgating modulation by membrane lipids remain to be discovered. 

The normal experimental technique is to scan rapidly through the velocity range and repeat this scan many times imtil data of the required accuracy has been accumulated. The Doppler motion is provided by an electromechanical drive system controlled by a servo -amplifier. Usually, the source is attached to the drive shaft and driven either in a saw-tooth or a triangular constant acceleration wave form. The transducer is coupled to a multichannel analyser operating in the multiscaler mode, and the servo-amplifier is controlled by the channel advance frequency. The dwell time in each channel, corresponding to a specific velocity increment, is 100 ps, and while the channel gate is open it accepts pulses from the detector. 

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