Molecular Basis for the Different Activation Kinetics of the Pacemaker Channels HCN2 and HCN4

Abstract

The pacemaker channels HCN2 and HCN4 have been identified in cardiac sino-atrial node cells. These channels differ considerably in several kinetic properties including the activation time constant ( act ), which is fast for HCN2 (144 ms at ؊140 mV) and slow for HCN4 (461 ms at ؊140 mV). Here, by analyzing HCN2/4 chimeras and mutants we identified single amino acid residues in transmembrane segments 1 and 2 and the connecting loop between S1 and S2 that are major determinants of this difference. Replacement of leucine 272 in S1 of HCN4 by the corresponding phenylalanine present in HCN2 decreased act of HCN4 to 149 ms. Conversely, activation of the fast channel HCN2 was decreased 3-fold upon the corresponding mutation of F221L in the S1 segment. Mutation of N291T and T293A in the linker between S1 and S2 of HCN4 shifted act to 275 ms. While residues 272, 291, and 293 of HCN4 affected the activation speed at basal conditions they had no obvious influence on the cAMP-dependent acceleration of activation kinetics. In contrast, mutation of I308M in S2 of HCN4 abolished the cAMP-dependent decrease in act . Surprisingly, this mutation also prevented the acceleration of channel activation observed after deletion of the C-terminal cAMP binding site. Taken together our results indicate that the speed of activation of the HCN4 channel is determined by structural elements present in the S1, S1-S2 linker, and the S2 segment.Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) 1 channels are thought to underlie the native pacemaker current, termed I f or I h , in the heart and brain where it contributes to the rhythmic activity of cardiac and neuronal pacemaker cells (1-4). All four members of the mammalian HCN channel gene family that have been cloned recently (5-9) share a highly preserved core region containing 6 transmembrane segments, including a voltage-sensing S4 segment and a pore region between S5 and S6, which is homologous to the S1-S6 region of voltage-gated potassium channels (K v ). The homology of the intracellular N and C termini within the HCN subtypes is less pronounced than for the core region with the exception of the highly conserved 120 amino acid long cyclic nucleotide binding domain (CNBD) starting about 80 amino acids downstream of S6.All four HCN channels are expressed in cardiac tissue (5, 10), but with some variations in the expression intensities among different species. HCN4 is highly expressed in the sino-atrial node. It is generally assumed that the slowly activating HCN4 contributes to the pacemaker activity and the modulation of the heart rate by ␤-adrenergic stimulation, whereas the less expressed, faster activated HCN2 and HCN1 may have additional functions such as maintaining the resting potential of pacemaker and other cells (11)(12)(13)(14).All four HCN channels are activated upon membrane hyperpolarization. Activation is voltage-dependent, i.e. the more hyperpolarized the membrane becomes, the faster the channels open. The HCN channels differ, however, greatly in their activ...

Point Mutation in the HCN4 Cardiac Ion Channel Pore Affecting Synthesis, Trafficking, and Functional Expression Is Associated With Familial Asymptomatic Sinus Bradycardia

“…11,12 However, the mutations in HCN4 itself may mimic vagal stimulation. 13 Mutant channels were found to be activated at more negative voltages 13,28,29 or to have reduced ability to be activated by cAMP 14 compared with the WT channels. As a result, they generated a smaller current during diastolic depolarization, resulting in a slowing of the heart rate.…”

Functional Expression of the Human HCN3 Channel

“…Electrophysiology-Electrophysiological recordings were performed as described (13). Currents were recorded in whole cell configuration at 23 Ϯ 1°C using a MultiClamp 700A amplifier and pClamp9 software (Axon Instruments/Molecular Devices).…”

“…However, the modulatory effect depends on the subtype (12,13); activation of HCN2 and HCN4 is accelerated much more by cAMP than that of HCN1 channels. Similarly, cAMP shifts the membrane potential for activation to a largerextentforHCN2andHCN4thanforHCN1.cAMPmodulationismediated by direct binding of cAMP to a cyclic nucleotide binding domain (CNBD) located in the intracellular C terminus, thereby releasing the inhibitory effect of this C terminus on channel activation (13)(14)(15).…”

Bradycardic and Proarrhythmic Properties of Sinus Node Inhibitors

“…Expression of HCN channels in HEK293 cells and voltage-clamp experiments were basically done as described previously (Stieber et al, 2003b). In brief, HEK293 cells were transfected with expression vectors encoding one of the human HCN channels using FuGENE6 transfection reagent (Roche, Mannheim, Germany) according to the manufacturer's instructions (transfectant/DNA ratio: 3:1, v/w) or by electroporation using a Bio-Rad system (Bio-Rad, Mü nchen, Germany) according to the manufacturer's instructions.…”