Integrated allosteric model of voltage gating of HCN channels

Hyperpolarization-activated (pacemaker) channels are dually gated by negati ve voltage and intracellular cAMP. Kinetics of native cardiac f-channels ar e not compatible with HII 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 WH gating, such as sigmoidal activa tion and deactivation, activation deviating from fixed power of an exponent ial, removal of activation "delay" by preconditioning hyperpolarization. Pr evious work on native channels has indicated that the shifting action of cA MP 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 al losteric model. We hypothesized that HCN channels are tetramers and that ea ch subunit comprises a Voltage sensor moving between "reluctant" and "willi ng" states, whereas voltage sensors are independently gated by voltage, cha nnel closed/open transitions occur allosterically These hypotheses led to a multistate scheme comprising five open and five closed channel states. Wie estimated model rate constants by fitting first activation delay curves an d single exponential time constant curves, and then individual activation/d eactivation traces. By simply using different sets of rate constants, the m odel accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the d ifferent kinetic properties of different isoforms. For example, faster kine tics 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 tha t reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus invol ves two processes: voltage sensor gating and allosteric opening/closing.