cardiac pacemaker currents. HCN4 was previously shown to localize to caveolae but how caveolae regulate the function of HCN4 is unknown. Caveolin-3, abundantly expressed in skeletal and cardiac muscles, is responsible for forming caveolae. We hypothesize that caveolin-3 regulates functions of HCN4. The caveolin-3 gene cloned from a human heart cDNA library using an RT-PCR technique was subcloned into pcDNA3. Wildtype (WT), a mutant (P104L) caveolin-3 and the vector pcDNA3 were transiently expressed in the HEK 293 cell lines stably expressing HCN4. The HCN4 currents were measured using whole-cell patch clamp technique. The P104L mutant has been shown to have a dominant-negative effect on WT caveolin and causes limb-girdle muscular dystrophy and cardiomyopathy. WT Cav3 but not P104L caused a 45% increase of HCN4 current density (WT: 42 14 pA/pF, P104L: 23 6 pA/pF, pcDNA3: 23 5, p 0.05). P104L caused a 2 fold increase in activation time constant for HCN4 current (ms: P104L: 2201 392 vs. WT: 1262 49 and pcDNA3: 1409 161, p 0.05), and shifted the voltage at 50% of the steady-state inactivation to more negative potential (P104L: -123 2 mV vs. WT: -115 1 mV and pcDNA3: -116 1 p 0.05). We conclude that the mutant P104L modulated gating kinetics of the HCN4 channel. Our data indicates that disruption of caveolar lipid rafts modifies gating kinetic properties of the cardiac pacemaker channels. The changes described would cause a reduction of pacemaker activity.