Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel

Henrietta Cserne Szappanos,Jakob Petereit,Padmapriya Muralidharan,Livia C Hool,A Harvey Millar,Evan Ingley

doi:10.1038/s41598-017-15087-0

Abstract

The “Fight or Flight” response is elicited by extrinsic stress and is necessary in many species for survival. The response involves activation of the β-adrenergic signalling pathway. Surprisingly the mechanisms have remained unresolved. Calcium influx through the cardiac L-type Ca2+ channel (Cav1.2) is absolutely required. Here we identify the functionally relevant site for PKA phosphorylation on the human cardiac L-type Ca2+ channel pore forming α1 subunit using a novel approach. We used a cell free system where we could assess direct effects of PKA on human purified channel protein function reconstituted in proteoliposomes. In addition to assessing open probability of channel protein we used semi-quantitative fluorescent phosphoprotein detection and MS/MS mass spectrometry analysis to demonstrate the PKA specificity of the site. Robust increases in frequency of channel openings were recorded after phosphorylation of the long and short N terminal isoforms and the channel protein with C terminus truncated at aa1504. A protein kinase A anchoring protein (AKAP) was not required. We find the novel PKA phosphorylation site at Ser1458 is in close proximity to the Repeat IV S6 region and induces a conformational change in the channel protein that is necessary and sufficient for increased calcium influx through the channel.

Highlights

The “Fight or Flight” response is elicited by extrinsic stress and is necessary in many species for survival
At the level of cardiomyocytes, β-adrenergic receptor (β-AR) stimulation leads to an increase in cyclic AMP production by adenylate cyclase coupled to GS proteins
A kinase anchoring proteins (AKAPs) is responsible for proper compartmentalization of protein kinase A (PKA) in cardiac myocytes[6,7,8,9], and binds to Cav1.2 channels via the leucine zipper motif in the DCT7,10 but in vivo studies have failed to prove a direct effect of AKAPs on cardiac contractility[11,12]

Summary

Introduction

The “Fight or Flight” response is elicited by extrinsic stress and is necessary in many species for survival. The distal carboxyl terminus (DCT) has been demonstrated to impose an autoinhibitory effect on channel function[4], contains the sites for calcium and calmodulin binding, and interaction with A kinase anchoring proteins (AKAPs)[5]. Serine 1700 and threonine 1704 in the proximal C terminal domain of cardiac Cav1.2 channels were identified as phosphorylation sites[20,21]. Both serine 1700 and threonine 1704 are conserved in the skeletal muscle Cav1.1 channels and serine 1575 and threonine 1579 have been identified as substrates for cAMP-dependent protein kinase. More recently it has been demonstrated that serine 1700 is not necessary for β-adrenergic responses in a mouse model[23] but is necessary for physiological regulation of the channel[24]

Methods

Results

Conclusion