The mammalian genome encodes four HCN channels (HCN1-4), which in response to hyperpolarisation activate an inward current (Ih) obviously important in generation and regulation of the heart rate. HCN4, the dominant HCN isoform in the sinoatrial node is associated with inherited sinus node dysfunction.

In a candidate gene approach, we identified different HCN4 mutations in two patients. Mutant and wild type (wt) HCN4 subunits were expressed in HEK293 cells and biophysical properties of mutant and wt channels were investigated by whole-cell patch-clamp technique.

In one of the index patients, brought to our attention by clinical symptoms of atrial fibrillation lacking bradycardia, we identified a G to C transversion in exon 4 of the HCN4 gene. Consequently, lysine 530 of the HCN4 channel is replaced by asparagine (HCN4 K530N). Analysis of the second index patient, attributed by symptoms of sinus node dysfunction with distinctive sinus bradicardia, revealed an insertion of 13 nucleotides in exon 6 of the HCN4 gene that caused a frame shift leading to a premature stop codon. Functional properties of HCN4 K530N channels revealed a positive 4 mV shift of reversal potential as compared to wt channels (Erev K530N: -18.38 0.5 vs Erev wt: -22.2 1.4 mV), resulting in a permeability ratio PNa/K of 0.29 0.01 and 0.22 0.03 for mutant and wt channel, respectively, which indicates a relatively enhanced permeability for Na+. However, it remained unclear whether and how this alteration influences the heart rhythm. Channels composed of HCN4 1932ins subunits carrying a truncated nucleotide binding domain were insensitive towards cAMP. In the presence of 10mM intracellular cAMP the time constant of activation (t) was significantly decreased for the wt channel (t at -140mV: 0.70 0.11 vs 0.48 0.05 s). For HCN4 1932ins channels no such effect was detected (t at -140mV: 0.53 0.04 vs 0.57 0.05 s). Additionally, intracellular cAMP shifted the potential of half maximal activation (V½) ~15 mV towards more positive potentials in wt channels (-87.5 3.3 to -73.2 1.8 mV), no shift was induced in HCN4 1932ins channels (-80.1 2.7 to -83.1 2.7 mV). Moreover, voltage-dependent activation in the absence of cAMP was shifted to intermediate values ranging between those found in wt channels in the presence and absence of cAMP. Hence, the HCN4 1932ins mutation might favour an impaired modulatory capacity of the heart rate. However patients, carrying the HCN4 1932ins mutation did not show signs of chronotropic incompetence in the clinical investigations, including a treadmill test.

Unlike the HCN4 K530N mutation, the HCN4 1932ins mutation carrying the truncated cyclic nucleotide binding domain is associated with clinical symptoms of sinus bradycardia. Interestingly, the lack of cAMP sensitivity of the mutated channel can obviously be compensated in vivo.