Related Experiment Video
Updated: Sep 30, 2026
Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes
Published on: January 10, 2011
Elimination of the BK(Ca) channel's high-affinity Ca(2+) sensitivity
Lin Bao1, Anne M Rapin, Ericka C Holmstrand
1Molecular Cardiology Research Institute, New England Medical Center, and the Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111, USA.
Abstract:
We report here a combination of site-directed mutations that eliminate the high-affinity Ca(2+) response of the large-conductance Ca(2+)-activated K(+) channel (BK(Ca)), leaving only a low-affinity response blocked by high concentrations of Mg(2+). Mutations at two sites are required, the "Ca(2+) bowl," which has been implicated previously in Ca(2+) binding, and M513, at the end of the channel's seventh hydrophobic segment. Energetic analyses of mutations at these positions, alone and in combination, argue that the BK(Ca) channel contains three types of Ca(2+) binding sites, one of low affinity that is Mg(2+) sensitive (as has been suggested previously) and two of higher affinity that have similar binding characteristics and contribute approximately equally to the power of Ca(2+) to influence channel opening. Estimates of the binding characteristics of the BK(Ca) channel's high-affinity Ca(2+)-binding sites are provided.
Insights
Site-directed mutations were used to eliminate the high-affinity calcium response of the large-conductance calcium-activated potassium channel (BK(Ca)). This study reveals BK(Ca) channels have three calcium binding sites, two with high affinity.
Area of Science:
- Molecular Biology
- Biophysics
- Ion Channel Physiology
Background:
- The large-conductance calcium-activated potassium channel (BK(Ca)) plays a crucial role in regulating cellular excitability.
- Understanding the precise mechanisms of calcium (Ca2+) activation in BK(Ca) channels is essential for interpreting its physiological functions.
Purpose of the Study:
- To elucidate the specific calcium binding sites and their contribution to the activation of BK(Ca) channels.
- To characterize the affinity and properties of different calcium binding sites within the BK(Ca) channel.
Main Methods:
- Site-directed mutagenesis was employed to alter specific residues within the BK(Ca) channel.
- Electrophysiological recordings were used to assess the functional consequences of these mutations on channel activity.
- Energetic analyses were performed to quantify the binding characteristics of calcium ions.
Main Results:
- Mutations at the "Ca(2+) bowl" and residue M513 collectively eliminated the high-affinity Ca(2+) response of BK(Ca) channels.
- A low-affinity, magnesium (Mg2+)-sensitive Ca(2+) response remained after mutagenesis.
- Energetic analyses suggest the presence of three distinct Ca(2+) binding sites: one low-affinity (Mg2+-sensitive) and two high-affinity sites with similar binding properties.
Conclusions:
- The BK(Ca) channel possesses multiple Ca(2+) binding sites that contribute to its activation.
- Two high-affinity Ca(2+) binding sites, contributing equally, are critical for Ca(2+) to effectively gate the channel.
- This study provides quantitative estimates for the binding characteristics of these high-affinity sites.
More Related Videos
11:08Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue
Published on: September 5, 2015
07:42Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates
Published on: October 18, 2018
Related Concept Videos
01:30Ligand-Gated Ion Channel Receptor: Gating Mechanism
01:16Calmodulin-dependent Signaling
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...