The long term goal of the present proposal is to define the physiological role of calcium-activated (KCa) and ATP-sensitive (KATP) K channels from coronary smooth muscle, by studying their biophysical characteristics, their response to vasoactive substances and their regulatory mechanism(s). The main hypothesis postulates that K+ channels may be modulated by vasoactive substances and intracellular messengers. Thus, their activity can control the resting potential and consequently the coronary tone. Two major types of K+ channels have been characterized in vascular smooth muscle, Ca2+-activated (KCa) and ATP-sensitive K+ (KATP) channels. However, little is known about the diversity and regulatory properties of these channels in coronary smooth muscle (CSM). Our initial findings indicate that CSM possesses KCa and KATP channels. KCa channels are abundant, have different functional properties, and may be inhibited by external application of vasoconstrictors (angiotensin II, AgII; thromboxane A2, TXA2), and activated by internally applied G proteins (Galphas). Thus, the key questions that I plan to address are: What are the biophysical and pharmacological characteristics of KCa and KATP channels from CSM? Are these channels spontaneously active in resting cells? What is the mechanism of inhibition of KATP channels by ATP? What is the mechanism of action of vasoactive substances on KCa channels, are G proteins involved? Are KATP channels modulated by vasoactive substances, as well? To answer these questions, I plan: 1) to study further the biophysical and pharmacological characteristics of KCa and KATP channels; 2) to characterize the action of AgII and TXA2 on KCa channels, and to explore potential actions of these vasoactive agents on KATP channels; 3) to determine the mechanism(s) of KCa channel modulation by AgII and TXA2 (direct ligand gating, direct G protein gating """"""""membrane delimited"""""""", and G protein mediated metabolic pathways leading to phosphorylation and/or arachidonic acid (AA) metabolism, will be explored); 4) to study if phosphorylation of dephosphorylation modify the actions of AgII and TXA2, and 5) to investigate if KCa and/or KATP channels are affected by vasorelaxants like AA and prostaglandin I2, and their mode of action. To accomplish the proposed aims, I will use a combined approach, which consists of studies using: a) the patch clamp technique (whole cell, cell attached and cell free patches) on single cells, and b) channels functionally reconstituted into bilayers. To perform patch clamp studies I have developed a single cell preparation from coronary muscle. In addition, I have succeeded in isolating a coronary plasma membrane enriched fraction which allowed to functionally reconstitute in bilayers KCa and KATP channels. These studies will help to understand how the coronary tone is regulated, how """"""""K+ channel openers"""""""" act to alleviate cardiovascular diseases, and probably to design therapeutical treatments that reduce coronary spasm responsible for myocardial ischemia and heart dysfunction.
