Molecular constituents encoding the cardiac pacemaker if currents

摘要

Cardiac pacemakers play critical biological functions in generating spontaneous and rhythmic action potentials.Defects in cardiac pacemaking can be life-threatening,leading to arrhythmia and sudden death.Although the hyperpolarization-activated,cyclic nucleotide-gated(HCN) channels play a prominent role in both cardiac and neuronal automaticity and excitability,multimerization of HCN1 -4 and the molecular constituents encoding the cardiac If currents have not been completely elucidated.Four members of the HCN family(HCN1-4) are identified in mammals as each HCN subunit contains a 6- transmembrane core domain and a cyclic nucleotide-binding domain(CNBD).In the myocardium,HCN4 is the most abundantly expressed transcript whereas HCN2 is readily detected in heart and brain tissues. Disruption of HCN4 causes early lethality in mice while mice with a targeted HCN2 display sinus dysrhythmia, which indicate that HCN4 and HCN2 both play important roles in cardiac pacemaking.Data from our group and others suggest that the heterolo-gously expressed HCN4 or HCN2 homomeric channels exhibit much more negative activation potentials relative to those recorded from cardiac cells,suggesting that a homomeric structure may not confer the cardiac If currents.We hypothesized that a HCN2/ HCN4 heteromeric channel is more likely to be the legitimate composition for cardiac HCN channels.The full-length HCN2 is estimated at 95-kDa,however, our results show that HCN2 in adult mouse hearts is a 60-kDa short form(designated as HCN2 C),which lacks the entire distal C-terminus including the cAMP regulatory domain.Thus,the cAMP-based regulation on the heterologous channel is possibly through the CNBD of HCN4.0ur results indicate that HCN2 C interacts with HCN4 in mouse hearts. We further show that a HCN2 C homomeric channel does not produce any currents.When HCN2 C is co-expressed with HCN4 in heterologous cells, pacemaking currents are recorded with the time- and voltage-dependent properties closely resemble those of endogenous cardiac If channels.These observations need to be confirmed in a cardiac system.In this research project,we aim at delineating the structure-function of HCN2 C/HCN4 channels, molecular generation of HCN2 C,and the complex regulation of HCN2 expression.Our long-term goal is to address the molecular structural components encoding cardiac HCN channels and develop novel therapeutic intervention strategies to improve cardiac arrhythmia that are related to defects in cardiac pacemaking.The objective for this proposal is to investigate the function/regulation of HCN2 C/HCN4 channels,and the molecular mechanisms that generate HCN2 C.Our central hypothesis is that HCN2 C is modified post-translationally,which co-assembles with HCN4 in adult mouse hearts as a het-eromeric channel.To accomplish our goals,we will undertake a combined approach of a HCN2 conditional knockout model,transgenic HCN2 C or full-length HCN2 "knock-in" mice,cardiac physiology, voltage clamping analysis and other biochemical assays to characterize HCN2 C/HCN4 channels.This research is expected to have a profound significance in clinical applications because of the high sequence homology between human and mouse HCN proteins. Our findings may provide new directions in designing pacemakers,identifying new drug targets and developing novel preventative interventions against cardiac arrhythmia.