Universität zu Köln

Neumaier, Felix ORCID: 0000-0002-6376-6391, Alpdogan, Serdar ORCID: 0000-0002-5188-9925, Hescheler, Juergen and Schneider, Toni ORCID: 0000-0003-2816-2696 (2018). Protein phosphorylation maintains the normal function of cloned human Ca(v)2.3 channels. J. Gen. Physiol., 150 (3). S. 491 - 511. NEW YORK: ROCKEFELLER UNIV PRESS. ISSN 1540-7748

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Abstract

R-type currents mediated by native and recombinant Ca(v)2.3 voltage-gated Ca2+ channels (VGCCs) exhibit facilitation (run-up) and subsequent decline (run-down) in whole-cell patch-clamp recordings. A better understanding of the two processes could provide insight into constitutive modulation of the channels in intact cells, but low expression levels and the need for pharmacological isolation have prevented investigations in native systems. Here, to circumvent these limitations, we use conventional and perforated-patch-clamp recordings in a recombinant expression system, which allows us to study the effects of cell dialysis in a reproducible manner. We show that the decline of currents carried by human Ca(v)2.3+beta(3) channel subunits during run-down is related to adenosine triphosphate (ATP) depletion, which reduces the number of functional channels and leads to a progressive shift of voltage-dependent gating to more negative potentials. Both effects can be counteracted by hydrolysable ATP, whose protective action is almost completely prevented by inhibition of serine/threonine but not tyrosine or lipid kinases. Protein kinase inhibition also mimics the effects of run-down in intact cells, reduces the peak current density, and hyperpolarizes the voltage dependence of gating. Together, our findings indicate that ATP promotes phosphorylation of either the channel or an associated protein, whereas dephosphorylation during cell dialysis results in run-down. These data also distinguish the effects of ATP on Ca(v)2.3 channels from those on other VGCCs because neither direct nucleotide binding nor PIP2 synthesis is required for protection from run-down. We conclude that protein phosphorylation is required for Ca(v)2.3 channel function and could directly influence the normal features of current carried by these channels. Curiously, some of our findings also point to a role for leupeptin-sensitive proteases in run-up and possibly ATP protection from run-down. As such, the present study provides a reliable baseline for further studies on Ca(v)2.3 channel regulation by protein kinases, phosphatases, and possibly proteases.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Neumaier, Felix UNSPECIFIED orcid.org/0000-0002-6376-6391 UNSPECIFIED Alpdogan, Serdar UNSPECIFIED orcid.org/0000-0002-5188-9925 UNSPECIFIED Hescheler, Juergen UNSPECIFIED UNSPECIFIED UNSPECIFIED Schneider, Toni UNSPECIFIED orcid.org/0000-0003-2816-2696 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-194457
DOI:	10.1085/jgp.201711880
Journal or Publication Title:	J. Gen. Physiol.
Volume:	150
Number:	3
Page Range:	S. 491 - 511
Date:	2018
Publisher:	ROCKEFELLER UNIV PRESS
Place of Publication:	NEW YORK
ISSN:	1540-7748
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Medicine > Physiologie und Pathophysiologie > Institut für Neurophysiologie
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language GATED CALCIUM-CHANNELS; PIG VENTRICULAR MYOCYTES; CARDIAC ALPHA(1) SUBUNIT; M-1 MUSCARINIC RECEPTORS; BOVINE CHROMAFFIN CELLS; C-TERMINAL TAIL; CA2+ CHANNELS; PATCH-CLAMP; PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE; PHOSPHOPROTEOMIC ANALYSIS Multiple languages Physiology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/19445