Abstract
The four subtypes (HCN1-4) of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in mammals show distinct activation and deactivation kinetics and cAMP sensitivities, which may lead to their distinct physiological roles. We previously elucidated that S1 and S1-S2 loop, and S6-cyclic nucleotide binding domain (CNBD) loop are important for activation kinetics by hyperpolarizing voltage steps. However, those regions affected deactivation kinetics much less than activation kinetics. Therefore, we investigated the structural basis for deactivation with the whole-cell patch clamp technique by using HCN1, HCN4, and chimeric channels in a mammalian expression system (COS-7). The deactivation time constant of HCN4 was about 7-fold longer than that of HCN1 when compared at 0 mV. In chimeras between HCN1 and HCN4, the replacement of the regions from the N-terminus to S3 and from S6 to CNBD affected the deactivation kinetics in a graded manner. The substitution of the region from S3 to S6 between HCN1 and HCN4, which includes the putative voltage sensor S4 and the pore region, did not affect the deactivation at all and affected the activation kinetics little. Furthermore, we investigated the effect of cAMP on deactivation kinetics. We especially focused on the single point mutation in S2, which was already reported to alter the effect of cAMP on activation kinetics. These results show that HCN channels modified not the region from S3 to S6 but the regions from N-terminus to S3 and from S6 to CNBD to create distinct subtypes in evolution. [Jpn J Physiol 54 Suppl:S136 (2004)]
