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A single histidine residue determines the pH sensitivity of the pacemaker channel HCN2
1Institut für Pharmakologie und Toxikologie der Technischen Universität München, Biedersteiner Str. 29, 80802 München, Germany.
Insights
Intracellular pH directly regulates HCN2 channels, a key component of heart and neuronal networks. A specific histidine residue (His-321) was identified as crucial for this pH sensitivity.
Area of Science:
- Molecular biology
- Cardiovascular physiology
- Neuroscience
Background:
- Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are crucial for cardiac and neuronal network rhythmicity.
- HCN channel activity is modulated by hormones, neurotransmitters, and potentially intracellular pH.
- HCN2 is a significant member of the HCN channel family.
Purpose of the Study:
- To investigate the direct modulation of HCN2 channels by intracellular pH.
- To identify the specific molecular determinants of pH sensitivity in HCN2 channels.
Main Methods:
- Site-directed mutagenesis of the HCN2 channel.
- Electrophysiological recordings to assess channel activity and voltage dependence.
- Analysis of proton and cAMP modulation in wild-type and mutant channels.
Main Results:
- Intracellular pH directly inhibits HCN2 channels by altering voltage dependence.
- A single histidine residue, His-321, at the S4-S5 linker boundary, is a major determinant of pH sensitivity.
- Mutations at His-321 abolished pH sensitivity while preserving cAMP modulation.
Conclusions:
- Intracellular pH is a direct regulator of HCN2 channel function within the physiological range.
- His-321 is a critical residue for pH sensing in HCN2 channels.
- pH regulation of HCN channels is likely a general mechanism conserved across the HCN family.
Abstract:
Hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels control the rhythmic activity of heart and neuronal networks. The activation of these channels is regulated in a complex manner by hormones and neurotransmitters. In addition it was suggested that the channels may be controlled by the pH of the cytosol. Here we demonstrate that HCN2, a member of the HCN channel family, is directly modulated by the intracellular pH in the physiological range. Protons inhibit HCN2 channels by shifting the voltage dependence of channel activation to more negative voltages. By using site-directed mutagenesis, we have identified a single histidine residue (His-321) localized at the boundary between the voltage-sensing S4 helix and the cytoplasmic S4-S5 linker of the channel that is a major determinant of pH sensitivity. Replacement of His-321 by either arginine, glutamine, or glutamate results in channels that are no longer sensitive to shifts in intracellular pH. In contrast, cAMP-mediated modulation is completely intact in mutant channels indicating that His-321 is not involved in the molecular mechanism that controls modulation of HCN channel activity by cyclic nucleotides. Because His-321 is conserved in all four HCN channels known so far, regulation by intracellular pH is likely to constitute a general feature of both cardiac and neuronal pacemaker channels.