Proteolytic activation of calcium channels: potential target for channel - directed therapeutics in arterial smooth muscle cells

The entry of Calcium Ions in excitable cells is the most ubiquitous electro-chemical trigger for vital processes in human physiology. Calcium entry is the common upstream message for the propagation of signals between brain cells (neurons), the contraction of the heart cells (cardiac myocytes), and the arterial blood pressure smooth muscle cells (SMCs). The excitation - evoked Calcium entry in all these cell types is controlled by a specialized class of protein complexes, called Voltage - Gated Calcium channels (VGCCs). VGCCs form a specialised pore in the otherwise impermeable cell membrane to regulate selectively the flux of Calcium among other charged ions into the excitable cells. Even minimal alterations from the normal VGCCs functions in the heart and blood vessels can lead to severe human diseases. As a consequence, VGCCs regulation is targeted by clinically important small - molecule drugs prescribed to reduce arterial blood pressure and treat cardiac dysfunctions. However, their wide distribution often results in off-target effects and there is a pressing need to find novel therapeutic approaches. To form a fully functional complex VGCCs requires co-assembly of several proteins (subunits) a central protein forming the calcium selective pore (called a1 subunit), and at least two auxiliary proteins (beta and a2d subunits) modulating VGCCs properties. Despite their pathological relevance, the structural and functional mechanisms underlying the regulation of VGCCs channels by a2d subunits remains poorly understood. Understanding of the molecular basis this regulation is of basic biological importance, and of medical importance, as a2d subunits are recognised as a potential route for developing novel VGCCs - directed therapeutic approaches.

I will pursue multidisciplinary approach by combining electrophysiological, biochemical, imaging and optical techniques to investigate the molecular mechanisms underlying the regulation of VGCCs by a2d1 subunits. I will focus on a novel physiological pathway for control VGCCs functionality determined by the enzymatic cleavage of a2d proteins. I will investigate both structural and functional aspects of this mechanism to determine how VGCCs ability to sense and respond to electrical signals is controlled by a2d by the novel enzymatic mechanism. In addition, I will assess the efficacy of novel VGCCs - directed inhibitors that act by supressing the enzymatic cleavage of a2d. I will examine the implications of targeting this pathway for the regulation of a2d1 in arterial SMCs.

I will benefit from building on my existing collaborations with several leading laboratories of complementary expertise in studies of electrical sensitivity of VGCCs using optical techniques (Professor Riccardo Olcese, UCLA) and structural biology (Dr. Mathew Gold, UCL). In addition, I will share expertise with specialists in studies of arterial SMCs in the host institution (Professors Iain Greenwood / Anthony Albert, SGUL) and a2d -knock out mice with Professor Annette Dolphin (UCL).

Our studies can break new ground in the understanding of the molecular basis of VGGCs regulation by a2d subunits that remains unresolved. By investigating the implications of this regulatory pathway for the physiological functions of a2d in arterial SMCs, we can provide rationale for development of future therapeutic approaches targeting pathological VGCCs dysfunctions in (such as hypertension). In addition to this, our experiments can provide conceptual advances applicable to other biological systems with different composition of VGCCs channel subtypes that are also regulated by a2d subunits (such as brain, and sensory neurons) relevant to different therapeutic areas (for example chronic pain).

The a2d1 subunits are key to the function of L-type Voltage-Gated Calcium channels (Cav1.2) in the regulation of cardiac contraction, vascular tone, and neuronal excitability. Abnormal Cav1.2 currents in arterial Smooth Muscle Cells (SMCs) are a hallmark of hypertension, but the involvement of a2d1 in the pathogenesis remain elusive. Despite their pathological relevance, the molecular mechanisms underlying Cav1.2 regulation by a2d1 are poorly understood.

I will pursue multidisciplinary approach by applying biochemical, imaging, optical and electrophysiological techniques to investigate the molecular basis of the regulation of Cav1.2 by a2d1 subunits. I will focus on a novel post-translational mechanism for control of the biological functions of a2d1 determined by the enzymatic cleavage into disulfide - linked alpha2 (a2) and delta (d) moieties.

I will undertake detailed structure-function analysis of the molecular properties of the linker domain between a2 and d (named proteolytic domain a2d-PD) that are of key importance for the biological functions of a2d1, and for the regulation by post-translational cleavage. I will build on my collaboration with crystallography experts in UCL to obtain the structure of the a2d-PD peptide.

I will assess the efficacy of synthetic peptides based on a2d-PD to act as novel Cav1.2 channels - directed inhibitors by supressing a2d1 cleavage. I will investigate the implications of targeting this pathway for the regulation of a2d1 in arterial Smooth Muscle Cells (SMCs), and as a potential route for development of novel Cav1.2 channels - directed therapeutics to reduce vascular resistance.

I will use an optical approach named Voltage Clamp - Fluorometry to obtain structural evidence of how voltage-sensing properties of Cav1.2 are controlled by the cleavage of a2d-PD in live cells.

The research will benefit from collaborations with experts in ion channel research in UCL, UCLA and in my host department of SGUL.