All mammals experience a slowdown of cardiac pacemaker rate with aging. The main mechanisms to explain that phenomenon are related to alterations in the ionic currents that underlie the diastolic depolarization phase of the action potential. We have previously reported that pacemaker cells from old mice have reduced L-type calcium currents. We further explore the mechanism underlying that reduction, testing cell hypertrophy and alteration in the scaffolding of L-type calcium channels as potential mechanisms. To test for cell hypertrophy, we combined immunostaining and high-resolution imaging to map the HCN4-positive pacemaker region of isolated upper heart explants from young and old mice. We compared cell length, width, and area between young and old cells. We also determined these morphological parameters in HCN4-positive enzymatically dissociated pacemaker cells. We found no significant difference in cell dimensions or area between ages, ruling out hypertrophy as a potential mechanism. We used mass spectrometry to identify expression changes in scaffolding proteins essential for calcium channel organization at the plasma membrane. Through this approach, we identified a large reduction of caveolin 3 as a possible mechanism. Caveolin is a protein essential to forming signaling microdomains between calcium channels and other proteins. Using western blotting, we confirmed a 50% reduction of caveolin 3 in isolated pacemaker tissues from old animals. Using proximity ligation assay and super-resolution microscopy, we showed altered recruitment of L-type calcium channels into caveolae. Our findings suggest that the age-associated decrease of L-type calcium current is caused by a reduced insertion of these channels in caveolae.

BK channels are potassium channels activated concomitantly by membrane depolarization and the elevation of intracellular calcium. We have previously shown that BK channels form clusters at the plasma membrane in heterologous cells and primary neurons, but the mechanism for their clustering is unknown. Our research seeks to discover important components that generate and maintain BK channel clusters. We hypothesize that membrane lipidic composition can be essential in BK clustering. Given the known role of PIP2 in increasing the activity of BK channels, we evaluated the role of PIP2 in their spatial organization. We expressed BK channels in a human cell line and assessed the organization in clusters using super-resolution microscopy and proximity ligation assay. We also measured ion channel mobility using fluorescence recovery after photobleaching. We expressed PIP5K and Ins5P to increase and decrease PIP2 levels, respectively. Preliminary experiments found that the expression of PIP5Kγ did not affect the mobility of single or cluster BK channels, but decreased density of channels at the plasma membrane.