The role of native cysteine residues in the oligomerization of KCNQ1 channels

Abstract

KCNQ1, the major component of the slow-delayed rectifier potassium channel, is responsible for repolarization of cardiac action potential. Mutations in this channel can lead to a variety of diseases, most notably long QT syndrome. It is currently unknown how many of these mutations change channel function and structure on a molecular level. Since tetramerization is key to proper function and structure of the channel, it is likely that mutations modify the stability of KCNQ1 oligomers. Presently, the C-terminal domain of KCNQ1 has been noted as the driving force for oligomer formation. However, truncated versions of this protein lacking the C-terminal domain still tetramerize. Therefore, we explored the role of native cysteine residues in a truncated construct of human KCNQ1, amino acids 100-370, by blocking potential interactions of cysteines with a nitroxide based spin label. Mobility of the spin labels was investigated with continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. The oligomerization state was examined by gel electrophoresis. The data provide information on tetramerization of human KCNQ1 without the C-terminal domain. Specifically, how blocking the side chains of native cysteines residues reduces oligomerization. A better understanding of tetramer formation could provide improved understanding of the molecular etiology of long QT syndrome and other diseases related to KCNQ1.

Keywords: EPR spectroscopy; KCNQ1; Kv channel; Oligomerization; SDS-PAGE.

Figure 1:

Cartoon depiction of monomeric KCNQ1 containing the voltage sensing domain (VSD), S1-S4; pore domain (PD), S5-S6; and the C-terminal domain, HA-HD. HC and HD are indicated in red.

Figure 2:

Representation of KCNQ1 used in this study, composed of the transmembrane domain (amino acids 100–370). Native cysteines are circled in purple. For portions of this study, C214 was changed to alanine (arrowhead). Helical regions are shaded in gray and labeled S0-S6. Modified from Dixit et al., 2022 [23].

Figure 3:

Schematic of the reaction between MTSL spin label and how it attaches to protein.

Figure 4:

Circular Dichroism spectra of all Q1 TMD variants in (0.09 mg/mL) 0.05% DPC and 50 mM phosphate buffer, pH 7.0. Q1 TMD WT −MTSL (blue), Q1 TMD WT +MTSL (black), Q1 TMD C214A −MTSL (green), and Q1 TMD C214A +MTSL (red).

Figure 5:

CW-EPR spectra of Q1 TMD WT (black) and Q1 TMD C214A (red) labeled with MTSL in 0.05% DPC and 50 mM phosphate buffer, pH 7.0.

Figure 6:

Silver Stained SDS-PAGE gel of all Q1 TMD variants. Lane 1: Page plus protein ladder (Fisher), Lane 2: Q1 TMD WT −MTSL, Lane 3: Q1 TMD WT +MTSL, Lane 4: Q1 TMD C214A −MTSL, Lane 5: Q1 TMD C214A +MTSL. All lanes have approximately 1 μg of protein. Monomeric Q1 TMD is approximately 24 kDa.