The Influence of Surface Charges on Quaternary Ammonium Block of Shaker K + Channels

Abstract

Block of K+ channels can be influenced by the ability of charged residues on the protein surface to accumulate cationic blocking ions to concentrations greater than those in bulk solution. We examined the ionic strength dependence of extracellular block of Shaker K+ channels by tetraethylammonium ions (TEA+) and by a trivalent quaternary ammonium ion, gallamine3+. Wild-type and mutant channels were expressed in Xenopus oocytes and currents recorded with the cut-open oocyte technique. Channel block by both compounds was substantially increased when the bathing electrolyte ionic strength was lowered, but with a much larger effect for trivalent gallamine. These data were quantitatively well described by a simple electrostatic model, accounting for accumulation of blocking ions near the pore of the channel by surface charges. The surface charge density of the wild-type channel consistent with the results was -0.1 e nm-2. Shaker channels with T449Y mutations have an increased affinity for both TEA and gallamine but the ionic strength dependence of block was described with the same surface charge density as wild-type channels. Much of the increased sensitivity of Shaker K+ channels to gallamine may be due to a larger local accumulation of the trivalent ion. The negative charge at position 431 contributes to the sensitivity of channels to TEA (MacKinnon & Yellen, 1990). A charge reversal mutation at this location had little effect on the ionic strength dependence of quaternary ammonium ion block, suggesting that the charge on this amino acid may directly affect binding affinity but not local ion accumulation.

Pharmacology and Surface Electrostatics of the K Channel Outer Pore Vestibule

“…The lines are fits of equation 1 to the data with K app values of 0.066 m M at normal and 0.017 m M at low ionic strength, a 3.9-fold increase in apparent gallamine affinity in the low-ionic strength solution. Similar results have been observed for gallamine block of Shaker K channels (Quinn & Begenisich, 2001). …”

“…As described above, TEA block of Shaker K channels is sensitive to solution ionic strength, and block by a trivalent TEA analog, gallamine, is even more sensitive (Quinn & Begenisich, 2001). These results demonstrate that negative charges on the surface of the channel contribute to an electrostatic potential that influences block by these charged molecules.…”

“…TEA block of some K channels is sensitive to ionic strength changes (MacKinnon et al, 1989; Quinn & Begenisich, 2001), indicating that surface electrostatics plays a role in the permeation and pharmacological properties of such channels. We showed here that this property is shared by Kv3.1 channels.…”

“…The pH-dependent TEA block of Kv1.1 channels is sensitive to changes in bulk solution ionic strength (Bretschneider et al, 1999). Finally, we have shown that TEA block of Shaker K channels is sensitive to bulk solution ionic strength and that block by a trivalent TEA analog (gallamine) is even more sensitive (Quinn & Begenisich, 2001). The results of these various studies suggest a role for surface electrostatics in quaternary ammonium block of K channels.…”

On the Mechanism of TBA Block of the TRPV1 Channel

“…Nevertheless, we can obtain an estimate of the expected effects of reduced ionic strength. The fixed surface charge near the external entrance of the pore of some channels has been estimated to be in the order of À0.06 to À0.1 e o /nm 2 (45,67). At our lowest Na 1 concentration, this would represent a concentrating factor for monovalent cations of three-to fivefold and this would be reduced to ;1.7fold at the higher Na 1 concentration.…”