Construction of a K Channel Hypersensitive to Polyunsaturated Fatty Acids

Abstract

Negatively charged polyunsaturated fatty acids (PUFAs) modulate the voltage dependence of several voltage-gated ion channels. The PUFAs are probably incorporated into the cell membrane close to the channel's voltage sensors wherein an electrostatic modulation occurs. The interaction site does not overlap with other known binding sites for toxins and drugs. For voltage-gated K channels, the PUFAs increase the open probability at negative membrane voltages thereby being potent modifiers of cellular excitability. However, in spite of the desirable effect of the PUFAs, they are probably no good pharmaceutical candidates due to their biological promiscuity. To find potent, and more specific small-molecule compounds, more knowledge about the modulation is needed. Our goal was to construct a K channel with very high sensitivity to PUFAs. We expressed the ShakerIR K channel in Xenopus oocytes and studied the ion currents with a two-electrode voltage clamp technique. Positive charges at the extracellular end of the voltage sensor S4 are expected to potentiate the effect of negatively charged PUFA molecules. Therefore we systematically introduced arginines, one by one, at long stretch of S4. The mutations with the largest effects were then combined in different ways. The most effective mutation, with respect to PUFA-induced shifts of the channel's voltage dependence, contained two extra arginines outside the positive charges of the wild-type S4. Introducing these two positively charged residues in combination with an altered pH from 7.4 to 9.0 increased the current about 100 times at negative voltages. This supersensitive channel can be instrumental in the search for medical drugs against hyperexcitability diseases such as cardiac arrhythmia, epilepsy, and pain.