The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I K1 ), which is important for maintenance of the cell resting membrane potential, and the sodium current (I Na ), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I K1 modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I K1 -I Na interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I K1 -I Na interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na V 1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na v 1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na v 1.5-Kir2.1 interactions. The reciprocal modulation between Na v 1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.reentry | scaffolding proteins | conduction velocity | protein trafficking I n the heart, the inward rectifying potassium current (I K1 ) is the major current responsible for the maintenance of the resting membrane potential (RMP), whereas the sodium current (I Na ) provides the largest fraction of the inward depolarizing current that flows during an action potential (1). It is well-known that a relationship exists between these two ionic currents that is crucial for proper cardiac electrical function; disruption of this balance results in changes in sodium channel availability, cell excitability, action potential duration, and conduction velocity (2). Accordingly, I K1 -I Na interactions are important in stabilizing and controlling the frequency of the electrical rotors that are responsible for the most dangerous cardiac arrhythmias, including ventricular tachycardia and fibrillation (3).Post synaptic density, discs large, and zonula occludens-1 (PDZ) domain proteins link different and in many cases, multiple proteins to macromolecular complexes through interactions with their various domains. More than 70 PDZ d...

Store operated calcium entry (SOCE) is an integral mechanism for calcium signaling and regulation within the cell. SOCE's key players are the Orai channels, calcium selective channel proteins located within the plasma membrane (PM), and the STIM proteins, Ca 2þ sensors located within the endoplasmic reticulum (ER) membrane. Upon ER Ca 2þ store depletion, STIM proteins form an active conformation that binds to and activates Orai channels. Structural and functional evidence indicate that Orai is allosterically activated by a chain of conformational changes originating from a physical interaction between Orai's C-terminus and STIM1's STIM/Orai activating region (SOAR). Although the mechanism of channel gating is unknown, it is presumed to be conserved amongst all three isoforms of Orai (Orai1, Orai2, Orai3). We chose to investigate Orai3, a less studied SOCE channel subtype shown to be upregulated in hypertrophied cardiomyocytes, and estrogen receptor-expressing breast cancers. To better understand Orai3's activation mechanism, we disrupted different intra-channel points of contact known to be important in Orai1's function using site directed mutagenesis. HEK triple Orai channel-knockout cells were transfected to overexpress our modified Orai3 channels and calcium entry was assessed using fluorescence microscopy. We found that the H109S and P254L mutations in Orai3 caused the channel to be constitutively active, similar to Orai1's corresponding mutations. Recently identified salt bridges connecting transmembrane regions of the protein were found to play an important role in transmitting the STIM1 binding signal to Orai1 channel gating. To test the function of these salt bridges in Orai3, we compared the activity of STIM1 activated channels to the H109S, and P254L constitutively active variants. Our results reveal an important role for the K60/E149 salt bridge in transmitting signals from Orai3's C-terminus to the channel pore.