External Tetraethylammonium As a Molecular Caliper for Sensing the Shape of the Outer Vestibule of Potassium Channels

Abstract

External tetraethylammonium (TEA+) blocked currents through Kv1.1 channels in a voltage-independent manner between 0 and 100 mV. Lowering extracellular pH (pHo) increased the Kd for TEA+ block. A histidine at position 355 in the Kv1.1 channel protein (homologous to Shaker 425) was responsible for this pH-dependent reduction of TEA+ sensitivity, since the TEA+ effect became independent of pHo after chemical modification of the Kv1.1 channel at H355 and in the H355G and H355K mutant Kv1.1 channels. The Kd values for TEA+ block of the two mutant channels (0.34 +/- 0.06 mM, n = 7 and 0.84 +/- 0. 09 mM, n = 13, respectively) were as expected for a vestibule containing either no or a total of four positive charges at position 355. In addition, the pH-dependent TEA+ effect in the wt Kv1.1 channel was sensitive to the ionic strength of the solution. All our observations are consistent with the idea that lowering pHo increased protonation of H355. This increase in positive charge at H355 will repel TEA+ electrostatically, resulting in a reduction of the effective [TEA+]o at the receptor site. From this reduction we can estimate the distance between TEA+ and each of the four histidines at position 355 to be approximately 10 A, assuming fourfold symmetry of the channel and assuming that TEA+ binds in the central axis of the pore. This determination of the dimensions of the outer vestibule of Kv1.1 channels confirms and extends earlier reports on K+ channels using crystal structure data as well as peptide toxin/channel interactions and points out a striking similarity between vestibules of Kv1.1 and KcsA channels.

Allosteric Block of KCa2 Channels by Apamin

“…TEA has been used as a molecular caliper to probe the inner pore of Kv channels (27). TEA could be used in the same way for K Ca 2 channels, but first, it had to be determined whether apamin and TEA bound to non-overlapping sites.…”

“…TEA can therefore be used as a "molecular caliper" to probe the dimensions of the inner pore under different conditions. Different sensitivities are proposed to indicate differences in pore structure and dynamics (26,27). Fig.…”

An Apamin- and Scyllatoxin-Insensitive Isoform of the Human SK3 Channel

“…We were intrigued that perhaps this novel isoform might refine our understanding of the pharmacological as well as physiological properties of the channel. To test whether the pore region of SK3 channels is affected by the 15 amino acid insertion, we tested different blockers that are known to bind at the outer vestibule, such as TEA ϩ (Bretschneider et al, 1999), d-tubocurarine, apamin (Ishii et al, 1997), and ScTX ). The S5-P-loop-S6 regions of SK2 and SK3 channels differ only at the residues Val485 and His521, which correspond to Val47 and Val95 in the KcsA channel and Tyr415 and Val451 in the Shaker potassium channel.…”

“…Interestingly, the TEA ϩ block was identical for both isoforms. For Kv1.1 channels, it has been shown that the TEA ϩ binding is close to the selectivity filter within the P-loop region (Yellen et al, 1991;Bretschneider et al, 1999). Because this region is not interrupted by the inserted amino acids in hSK3_ex4, the TEA ϩ binding sites of hSK3_ex4 would be assumed not to be affected by the insertion.…”

Novel Neuroprotective K+Channel Inhibitor Identified by High-Throughput Screening in Yeast

“…Nothing is known about modulation of K ϩ channels specifically by norbornane and similar groups. Catechol in concentrations above 0.1 mM has been used widely to inhibit native K ϩ currents (Ito and Maeno, 1986;Erdelyi and Such, 1988;Kuenzi and Dale, 1998;Bretschneider et al, 1999;Hess and El Manira, 2001); however, the mechanism of its action remains unknown. Our data on catechol inhibition of cloned Kv channels indicate that the site of catechol action is different from that of 48F10, suggesting that 48F10 may constitute a novel pharmacophore for K ϩ channels (manuscript in preparation).…”