| Abstract: | ![]()
Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties.Voltage-gated calcium channels (L-, N-, P/Q-, R-, and T-types) mediate calcium entry in many different cell types in response to membrane depolarization and action potentials. Calcium influx through these channels serves as an important second messenger of electrical signaling, initiating a variety of cellular events and physiological functions (1, 2). Among the family of voltage-gated calcium channels, T-type calcium channels (Cav3 family) have unique electrophysiological properties, because they display low voltage-activated calcium currents with rapid activation/inactivation kinetics. In neurons, small changes in the membrane potential near the resting potential can activate T-type channels, favoring further membrane depolarization and repetitive firing of action potentials (3–5). These unique gating properties of T-type channels make them important in many different processes, including neuronal spontaneous firing and pacemaker activities, rebound burst firing, sleep rhythms, sensory processing, and neuronal differentiation, as well as in pathological conditions, such as epilepsy and neuropathic pain (6).To properly assure this plurality of physiological functions, a tight control of T-type calcium channels is necessary. An important regulatory mechanism is phosphorylation, the fastest and most frequent posttranslational modification for a protein. Ion channels, especially voltage-gated channels, are critically regulated by phosphorylation. Voltage-dependent sodium and potassium channels have been shown to be the target of multiple phosphorylation events, regulating different channel functions and being involved in pathological states, like epilepsy (7–11). Also, several studies have shown the crucial role of the L-type/Cav1 phosphorylation in important physiological functions, like the fight or flight response (12–15).Regarding T-type channels, phosphorylation remains poorly understood. There are three Cav3 pore-forming proteins (Cav3.1, Cav3.2, and Cav3.3 subunits) all displaying typical properties of T-type channels when expressed in heterologous cell systems (3, 16). Among them, the Cav3.2 channel seems to be particularly sensitive to various types of regulation, including phosphorylation. To date, several serine/threonine kinases, like PKA, PKC, or CamKII, have been shown to regulate Cav3.2 activity (reviewed in refs. 17–20); however, in most cases, this regulation is tissue-dependent, and little is still known about its molecular basis. Cav3.2 channels bear more than 100 multiple intracellular serine and threonine residues that are predicted to be phosphorylated by common prediction algorithms, like NetPhos2.0 (21). However, which of these residues are actually phosphorylated and what functional impact this phosphorylation will have remain to be determined.In this study, we investigate the phosphorylation pattern of the Cav3.2 isoform of the T-type channels and its role in Cav3.2 channel properties. Using an MS approach, we have established the first, to our knowledge, phosphorylation map of the Cav3.2 channel in a mammalian brain and a human cell line. Then, by using alkaline phosphatase (AP) and dephosphomimetic mutants in patch-clamp experiments, we reveal the importance of phosphorylation in modulating Cav3.2 gating properties. We have also identified a phosphorylation hot spot situated in the loop connecting domains I and II of the channel that plays a crucial role in this regulation. Altogether, this study provides important insights regarding how phosphorylation regulates Cav3.2 channels. |
