Integrated Allosteric Model of Voltage Gating of Hcn Channels

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.

Interaction of the Pacemaker Channel HCN1 with Filamin A

“…Both activation and deactivation time constants were much slower (1.4 -3-fold) in A7 cells than in M2 cells. Both the activation curve and the activation/deactivation time constant curves in A7 cells were similar to those measured previously for mHCN1 expressed in human embryonic kidney 293 cells (5).…”

“…Deactivation traces were recorded by fully activating the current at Ϫ135 mV and then stepping to the range of Ϫ55 to 45 mV for a time long enough to reach steady-state. Time constants were calculated by fitting activation/deactivation traces to a monoexponential function after an initial delay (5), and the values were averaged and plotted against voltage. Conductances were normalized to cell capacitance, measured by a specific 10-mV step protocol.…”

“…The molecular correlates of f/h channels are the hyperpolarizationactivated cyclic nucleotide-gated channels, of which four isoforms (HCN1-4) are known. When expressed alone in heterologous systems, HCN channels elicit currents whose properties are qualitatively similar to those of native I f /I h currents, although they differ quantitatively in kinetics and cAMP sensitivity (4,5).…”

Allosteric gating mechanism underlies the flexible gating of KCNQ1 potassium channels

“…Although this is the only member of the Kv channel family to be shown to gate in this way, the related BK channel does use its voltage sensors as allosteric regulators of the opening transition, along with binding of Ca 2+ . HCN channels, close relatives to Kv channels, also gate by an allosteric mechanism (25,26).…”

HCN4-like immunoreactivity in rat retinal ganglion cells

“…This would predict that the contribution of I h to resting membrane potential and resistance (Lee and Ishida, 2007) and to rebound excitation (Tabata and Ishida, 1996;Lee et al, 2003;Lee and Ishida, 2007) is increased by conditions that elevate intracellular cAMP levels (Vaquero et al, 2001;Nir et al, 2002;Dunn et al, 2006;Mills et al 2007). However, properties of the net current are shaped by the presence of other HCN isoforms (Franz et al, 2000;Santoro et al, 2000;Altomare et al, 2001;Chen et al, 2001). It would therefore be natural for future studies to test the possibility that HCN4 channels co-localize with other HCN channels in retinal ganglion cells.…”