Functional Expression of the Human HCN3 Channel

Abstract

Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels underlie the inward pacemaker current, termed I f /I h , in a variety of tissues. Many details are known for the HCN subtypes 1, 2, and 4. We now successfully cloned the cDNA for HCN3 from human brain and compared the electrophysiological properties of hHCN3 to the other three HCN subtypes. Overexpression of human HCN3 channels in HEK293 cells resulted in a functional channel protein. Similar to hHCN2 channels, hHCN3 channels are activated with a rather slow time constant of 1244 ؎ 526 ms at ؊100 mV, which is a greater time constant than that of HCN1 but a smaller one than that of HCN4 channels. The membrane potential for half-maximal activation V1 ⁄ 2 was ؊77 ؎ 5.4 mV, and the reversal potential E rev was ؊20.5 ؎ 4 mV, resulting in a permeability ratio P Na /P K of 0.3. Like all other HCNs, hHCN3 was inhibited rapidly and reversibly by extracellular cesium and slowly and irreversibly by extracellular applied ZD7288. Surprisingly, the human HCN3 channel was not modulated by intracellular cAMP, a hallmark of the other known HCN channels. Sequence comparison revealed >80% homology of the transmembrane segments, the pore region, and the cyclic nucleotide binding domain of hHCN3 with the other HCN channels. The missing response to cAMP distinguishes human HCN3 from both the well cAMP responding HCN subtypes 2 and 4 and the weak responding subtype 1.Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) 2 channels are thought to underlie the native pacemaker current, termed I f or I h , in the heart and brain where these channels contribute to the control of the resting membrane potential and to the rhythmic activity of excitable cells (1-4). Four mammalian genes encoding HCN channels have been identified to date (5-7). Electrophysiological studies of the three expressed HCN (HCN1, -2, and -4) channels showed (8) that each channel is activated voltage-dependent upon membrane hyperpolarization with distinct activation kinetics (5, 9, 10). Activation of the HCN channels is directly modulated by cyclic nucleotides (11). 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 (13-15).The functional expression of HCN3 channels proved to be difficult. Expression of the murine HCN3 in HEK293 cells or similar systems resulted in very small and unstable currents. Only recently, expression of the murine HCN3 channel was improved by using a lentiviral-based approach (16). Thus, information about the basic biophysical and pharmacological properties of HCN3 channels, in comparison to the other HCN channels, is limited (5,8,16,17). Esp...

PIP2-Mediated HCN3 Channel Gating Is Crucial for Rhythmic Burst Firing in Thalamic Intergeniculate Leaflet Neurons

“…1, 2), consistent with the prominent HCN2 expression in these cells (Notomi and Shigemoto, 2004). These data are consistent with the conclusion that cAMP insensitivity distinguishes HCN3 from the other HCN subunits (Stieber et al, 2005), and lead us to conclude that that HCN3 channels are the source of I h in IGL neurons.…”

“…Third, HCN2/3 heteromers do not appear to exist (Much et al, 2003). Thus, our I h data indicate that no cAMP-sensitive homomeric HCN2 channels are involved, and are in keeping with the observed biophysical properties of recombinant HCN3 channels, which feature slow activation kinetics and no facilitatory gating effect during cAMP application (Mistrík et al, 2005;Stieber et al, 2005).…”

“…Functional expression of HCN3 in IGL neurons is correlated with biophysical properties of I h HCN3 is variably expressed in both the rodent Notomi and Shigemoto, 2004;Mistrík et al, 2005) and human (Stieber et al, 2005) brain. We detected very dense HCN3-IR in somata of IGL neurons from HCN2 Ϫ/Ϫ and HCN2 ϩ/ϩ mice as well as rats.…”

“…cAMP insensitivity of I h in IGL, but not vLGN, neurons Distinct cAMP response patterns have been found for HCN channel subtypes and proven useful for identification and characterization of I h generated by HCN subunits (Mistrík et al, 2005;Stieber et al, 2005) [for review, see Gauss and Seifert (2000) and Robinson and Siegelbaum (2003)]. Consequently, we tested cAMP modulation of I h in IGL neurons.…”

“…Ligand-mediated gating constitutes a major pathway for native channels, as evidenced by a large hyperpolarizing shift (Ϫ40 to Ϫ60 mV) in voltage dependence due to loss of intracellular regulatory factors, including cAMP and phosphatidylinositol-4,5-bisphosphate (PIP 2 ) (Pian et al, 2006;Zolles et al, 2006); cAMP and PIP 2 facilitate gating for three HCN (1, 2, and 4) subunits. Gating of recombinant HCN3 channels, however, is not enhanced by cyclic nucleotides (Mistrík et al, 2005;Stieber et al, 2005), and their sensitivity to PIP 2 is unknown. Although native "HCN3 currents" are reported for the rat entopeduncular nucleus (Meurers et al, 2009), strong expression of HCN2 in this nucleus (Notomi and Shigemoto, 2004) makes it difficult to identify properties of natively expressed HCN3 channels.…”