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Integrated allosteric model of voltage gating of HCN channels
C Altomare1, A Bucchi, E Camatini
1Dipartimento di Fisiologia e Biochimica Generali, via Celoria 26, and INFM-Unità Milano Università, via Celoria 16, 20133 Milano, Italy.
Insights
Hyperpolarization-activated channels, crucial for heart rhythm, are gated by voltage and cAMP. A new allosteric model explains their complex voltage-dependent gating, revealing distinct properties across HCN channel isoforms.
Area of Science:
- Biophysics
- Molecular and Cellular Physiology
Background:
- Hyperpolarization-activated (pacemaker) channels, including HCN channels, are essential for cardiac rhythm.
- Their gating kinetics do not conform to the Hodgkin-Huxley model and necessitate multistate models.
- Previous studies suggested an allosteric mechanism for cAMP modulation of channel open probability.
Purpose of the Study:
- To investigate if an allosteric model can explain the voltage-dependent gating of hyperpolarization-activated channels.
- To propose a structural and mechanistic hypothesis for HCN channel gating.
Main Methods:
- Developed a multistate allosteric model with five open and five closed states, incorporating voltage sensors and allosteric transitions.
- Estimated model rate constants by fitting experimental data, including activation delay and deactivation traces.
- Applied the model to HCN1, HCN2, and HCN4 isoforms.
Main Results:
- The allosteric model successfully reproduced the complex voltage-dependent gating of all three HCN isoforms.
- The model explains isoform-specific kinetics, such as faster gating in HCN1 compared to HCN2/HCN4.
- It also accounts for experimental observations regarding voltage sensor charge mutations.
Conclusions:
- HCN channel voltage gating involves a two-step process: independent voltage sensor activation and allosteric channel opening/closing.
- This allosteric framework provides a unified explanation for the gating properties of different HCN channel isoforms.
- The findings offer insights into the molecular mechanisms underlying cardiac pacemaker channel function.
Abstract:
Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.