Pflügers Arch – Eur J Physiol (2000) 439:618–626 Digital Object Identifier (DOI) 10.1007/s004249900225
O R I G I N A L A RT I C L E
Anna Moroni · Andrea Barbuti · Claudia Altomare Carlo Viscomi · Julie Morgan · Mirko Baruscotti Dario DiFrancesco
Kinetic and ionic properties of the human HCN2 pacemaker channel Received: 11 October 1999 / Received after revision: 19 November 1999 / Accepted: 22 November 1999 / Published online: 12 January 2000 © Springer-Verlag 2000
Abstract Human cDNA coding for the hyperpolarization-activated “pacemaker” channel HCN2 was expressed in Phoenix cells and yielded an inward current (IhHCN2) activated on hyperpolarization. The average IhHCN2 was half-activated at –83.1 mV and its kinetics could be described by second-order Hodgkin–Huxley gating. The time constant curve was bell-shaped and peaked at –82.2 mV. With 115 mM external Na+ and 30 mM external K+, IhHCN2 reversed at –17.1 mV, and had a mean conductance of 5.6 nS. Reducing the external K+ or Na+ concentration led to a concentration-dependent reduction of the IhHCN2 conductance and to a hyperpolarizing shift of reversal potential. External Cs+ ions (5 mM) blocked IhHCN2 in a voltage-dependent way according to a Woodhull-type block model, at an electrical distance of 0.66 from the external membrane surface, and with a dissociation constant of 15 mM at 0 mV. Increasing cytoplasmic cAMP using forskolin increased IhHCN2 by shifting the current activation curve to more positive voltages (11.7 mV). Exposure of the intracellular side of inside-out macro-patches to cAMP led to a depolarizing shift of the channel open probability curve (15.2 mV with 10 µM cAMP). These results indicate that although hHCN2 channels share several properties with native cardiac f-channels, differences also exist in permeability and block properties, suggesting that native channels may not be composed simply of homomeric constructs. Key words cAMP-activated channels · Cardiac pacemaker · HCN clones · Hyperpolarization-activated channels · If current
A. Moroni · A. Barbuti · C. Altomare · C. Viscomi · J. Morgan M. Baruscotti · D. DiFrancesco (✉) Università di Milano, Dipartimento di Fisiologia e Biochimica Generali, via Celoria 26, 20133 Milano, Italy e-mail:
[email protected] Tel.: +39-2-70644603, Fax: +39-2-70632884
Introduction The properties of the pacemaker (f/h) current in many cell types have been established on the basis of several detailed studies over the last two decades [9, 28]. Much of the present knowledge of the specific features of these channels comes from studies of the cardiac sino-atrial node (SAN) region of the rabbit [10]. Recently, a family of hyperpolarization-activated, cyclic-nucleotide-gated channels (HCN family, HCN1 to HCN4) was cloned, whose expression yields currents with features analogous to those of native f/h channels [4]. This has prompted investigation aimed at determining the correspondence between HCN isoforms and native pacemaker channels. To date, functional expression of HCN1, HCN2 and HCN4 has been reported [23, 25, 26, 30, 31, 33]. The functional properties of the various isoforms are not homogeneous. For example, mHCN1 is essentially insensitive to internal cAMP [31], which contrasts with the higher sensitivity of both HCN2 and HCN4 [23, 25, 26]. The voltage activation range is also variable. Whereas mHCN1 and mHCN2 have half-activation voltages (V1/2) of –100 and –103 mV, respectively [25, 31], V1/2 values of –97 mV for hHCN2, –109 mV for hHCN4 [26] and –87 mV for rbHCN4 [23] have been reported. This shows that some variability is also found for the same isoform in different species. Finally, differences exist among isoforms in the kinetics of activation. For example, hHCN4 activates more slowly than hHCN2 [26, 33], and very slow activation has been observed for mHCN2 in comparison to mHCN1 [30]. These data suggest that the functional variability found in native hyperpolarization-activated currents [9, 28] may result from a non-uniform tissue distribution of channel isoforms with different functional features, or from a heteromultimeric composition of native channels. It is therefore important to couple analysis of the functional expression of the different isoforms to the investigation of their tissue distribution. More than one isoform is expressed in cardiac tissue. In mouse, HCN2 is expressed in atrium, ventricle and SAN [31]. In rabbit,
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RNase protection assays and in situ hybridization data show that only trace amounts of HCN2 are detected in SAN and ventricle [32], while major expression of both HCN4 and HCN1 isoforms is found [27, 32]. Since the HCN4 isoform is highly expressed in rabbit SAN, Ishii et al. [23] proposed that it is a major component of the native f-channel there. Less detailed information is available concerning isoform distribution in human heart. Northern blot analysis indicates the presence of similar amounts of HCN2 and HCN4, but not HCN1, in the heart [25, 31]. This, together with original observations of the electrophysiological characteristics of hHCN2 [26, 33], suggests that this isoform may contribute fundamentally to the functional properties of native cardiac pacemaker channels in humans. In this work, we have extended analysis of the functional properties of hHCN2 through expression in Phoenix cells. We report here that the major kinetic and ionic features of the hHCN2 channel, and their dependence on intracellular cAMP are similar to those of native f-channels; on the other hand, differences are found in the dependence upon extracellular ions and in the Cs+induced block, which appears to occur at the same membrane “electrical” distance, but with a much lower efficiency, than with f-channels.
Materials and methods Molecular biology We identified the human HCN2 gene and deduced the coding sequence of the protein as previously described [33]. To express human HCN2 we used a minigene which was derived partially from genomic clones and partially from mRNAs retrotranscribed from human cerebellum. Briefly, we retrieved from LLNL (USA) two contiguous but not overlapping cosmids, F18382 (AC005559, AC005577) and R33683 (AC00 4449). The two cosmids included the 5’ and 3’ portions of the human HCN2 gene, respectively, and we covered the gap between them with a fragment of HCN2 DNA previously retrieved by reverse transcriptase polymerase chain reaction (RT-PCR) from human cerebellum. For functional expression, we subcloned the construct into the expression vector pcDNA3.1 (+) (Invitrogen), which carries a cytomegalovirus (CMV) strong promoter. This construct was cotransfected with a plasmid containing green fluorescent protein (GFP) into modified HEK293 (Phoenix) cells [24] for expression. The cells were transiently transfected with a mixture of the two vectors following the calcium phosphate transfection protocol [29]. For each 35 mm Petri dish we used 7 µg HCN2-containing and 3.5 µg GFP-containing plasmid. Phoenix cells were cultured in Dulbecco’s modified essential medium (GIBCO BRL) supplemented with 10% fetal calf serum (GIBCO BRL) and antibiotics (Sigma). Visual inspection of transfected cells 24 h after transfection indicated a transfection efficiency of about 20%. Electrophysiology Following transfection, cells were incubated at 37°C in 5% CO2 for up to 4–7 days. In pilot experiments we noticed robust expression of hHCN2 after at least 24 h from transfection. Plastic Petri dishes were placed under the stage of an inverted microscope, and GFP-fluorescent cells selected by visual inspection were analysed using the patch-clamp technique. The patch-clamp methods were
the same as previously reported in a study of isolated cardiac myocytes [15]. Cells were superfused with a Tyrode solution containing (mM): NaCl, 110; KCl, 30; CaCl2, 1.8; MgCl2, 1; D-glucose, 5.5; HEPES-NaOH, 5 (pH=7.4). Control and test solutions were delivered via a perfusion pipette allowing rapid (
