threonine PKC sites of the Cav2.3α1 subunits

Accepted Manuscript Stimulatory and inhibitory effects of PKC isozymes are mediated by serine/threonine PKC sites of the Cav2.3α1 subunits Senthilkumar Rajagopal, Brittney K. Burton, Blanche L. Fields, India O. El, Ganesan L. Kamatchi PII:

S0003-9861(16)30576-8

DOI:

10.1016/j.abb.2017.04.002

Reference:

YABBI 7457

To appear in:

Archives of Biochemistry and Biophysics

Received Date: 16 December 2016 Revised Date:

8 March 2017

Accepted Date: 3 April 2017

Please cite this article as: S. Rajagopal, B.K. Burton, B.L. Fields, I.O. El, G.L. Kamatchi, Stimulatory and inhibitory effects of PKC isozymes are mediated by serine/threonine PKC sites of the Cav2.3α1 subunits, Archives of Biochemistry and Biophysics (2017), doi: 10.1016/j.abb.2017.04.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1 Stimulatory and Inhibitory Effects of PKC isozymes are Mediated by Serine/Threonine PKC Sites of the Cav2.3α1 subunits

Department of Biology, Norfolk State University, Norfolk, Virginia 23504.

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Senthilkumar Rajagopal1, Brittney K. Burtona, Blanche L. Fieldsa,2, India O. Ela, Ganesan. L. Kamatchia,∗

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Corresponding Author at: Department of Biology, Norfolk State University,700 Park Avenue, Norfolk, VA 23504 E-mail address: [email protected]

Present Address: Department of Biochemistry, Rayalseema University, Kurnool, AP, INDIA Present Address: Department of Biological Sciences, 600 Fairchild Center, Columbia University, New York, NY 10027 2

ACCEPTED MANUSCRIPT 2

ABSTRACT Protein kinase C (PKC) isozymes modulate voltage-gated calcium (Cav) currents through Cav2.2

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and Cav2.3 channels by targeting serine/threonine (Ser/Thr) phosphorylation sites of Cavα1 subunits. Stimulatory (Thr-422, Ser-2108 and Ser-2132) and inhibitory (Ser-425) sites were identified in the Cav2.2α1 subunits to PKCs βII and ε. In the current study, we investigated if the

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homologous sites of Cav2.3α1 subunits (stimulatory: Thr-365, Ser-1995 and Ser-2011; inhibitory:

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Ser-369) behaved in similar manner. Several Ala and Asp mutants were constructed in Cav2.3α1 subunits in such a way that the Ser/Thr sites can be examined in isolation. These mutants or WT Cav2.3α1 along with auxiliary β1b and α2/δ subunits were expressed in Xenopus oocytes and the effects of PKCs βII and ε studied on the barium current (IBa). Among these sites, stimulatory Thr-365 and Ser-1995 and inhibitory Ser-369 behaved similar to their homologs in Cav2.2α1

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subunits. Furthermore PKCs produced neither stimulation nor inhibition when stimulatory Thr365 or Ser-1995 and inhibitory Ser-369 were present together. However, the PKCs potentiated the IBa when two stimulatory sites, Thr-365 and Ser-1995 were present together, thus overcoming

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the inhibitory effect of Ser-369. Taken together net PKC effect may be the difference between

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the responses of the stimulatory and inhibitory sites.

Keywords: Barium current, Cav channels, Isozymes, Regulation, Xenopus oocytes, protein interaction

ACCEPTED MANUSCRIPT 3 Highlights: Stimulatory and inhibitory sites were found in Cav2.3α1 subunits for PKCs βII and ε

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Thr-365 and Ser-1995 were stimulatory and Ser-369 was inhibitory to PKCs βII and ε

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Stimulatory and inhibitory PKC sites are homologous in Cav2.3α1 and Cav2.2α1 subunits

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Net PKC effect is the difference in responses of stimulatory and inhibitory sites

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ACCEPTED MANUSCRIPT 4 Abbreviations

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IBa - Basal barium current, Cav - Calcium channels, PKC - Protein kinase C, MCh Acetyl βmethylcholine, PMA- Phorbol-12-myristate, 13-acetate, V1/2 - Half inactivation

ACCEPTED MANUSCRIPT 5 1.

Introduction Cav channels are multi-subunit protein complexes consisting of a pore-forming α1 subunit

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and smaller auxiliary β, α2/δ and γ subunits. These channels are classified into Cav1 (L-type), Cav2 (2.1 P/Q-type, 2.2 N-type and 2.3 R-type) and Cav3 (T-type) families based primarily on the sequence homology of the α1 subunits [1, 2]. In general, members of Cav2 family are

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involved in neurotransmitter release and undergo modulation by second messengers such as Gprotein βγ subunits, calmodulin and PKC isozymes [3-7]. For example, Cav2.2 and 2.3 currents

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were potentiated by phorbol-12-myristate, 13-acetate (PMA), an activator of PKCs βII and ε [8, 9] whereas Cav2.1 currents were not affected [7]. Similarly, when challenged with acetyl βmethylcholine (MCh), an agonist of muscarinic M1 receptors and an activator of PKCα [9], Cav2.3 channels alone were potentiated [5]. In addition, PKC activation by the influx of Ca2+ also

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augmented Cav2.3 currents [10]. Further it has been found by some that Cav2.3 currents undergo dual modulation by muscarinic receptor activation; that is inhibition by G-protein βγ subunits and stimulation through PKC [11]. Dual regulation of Cav2.3 currents by M1 receptors was also

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found by others; however, suppression of the currents was mediated by the depletion of

[12].

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membrane phosphatidylinositol 4, 5-bisphosphate and potentiation through the activation of PKC

Cavα1 subunits, esp., their intracellular segments are the target of PKC isozymes. Ser/Thr

PKC phosphorylation sites responsible for PMA- or M1 receptor-induced modulation of Cav2.2 and Cav2.3 channels were identified by several laboratories including ours. For example, Thr422, Ser-425, Ser-2108 and Ser-2132 of Cav2.2α1 subunits are the site of action of PMA, PKCβII or ε isozymes [13-15]. Similarly in the Cav2.3α1 subunits, Ser-888, Ser-892, Ser-894 and Ser-

ACCEPTED MANUSCRIPT 6 1987 are identified as MCh-sensitive [5] and Thr-365, Ser-369, Ser-1995 and Ser-2011 as PMAsensitive PKC targets [4]. Furthermore it has been recognized that the Ser/Thr of these subunits can mediate the stimulatory or inhibitory actions of PKC isozymes. Among the four PKC sites

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that were identified in the Cav2.2α1 subunit, Thr-422, Ser-2108 and Ser-2132 mediated the stimulatory actions and Ser-425 facilitated the inhibitory effects of PKCs [14].

Based on this background, in this study, we characterized the PMA-sensitive sites of

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Cav2.3α1 subunits since Thr-365, Ser-369, Ser-1995 and Ser-2011 are homologous to Thr-422, Ser-425, Ser-2108 and Ser-2132 of Cav2.2α1 subunits respectively [5]. WT and Ser/Thr→

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alanine (Ala) or aspartate (Asp) mutant constructs of Cav2.3α1 subunits were expressed along with β1b and α2/δ in Xenopus oocytes and the resultant currents were challenged with PKC isozymes βII or ε by injecting them into the oocytes. The results revealed that the homologous Ser/Thr sites of Cav2.3α1 subunits behaved similar to that of Cav2.2α1 subunits. In addition the

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relative dominance of these sites, based on the various mutant constructs, was investigated. Materials and Methods

2.1 Harvesting of Oocytes and cDNA injection

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Mature female Xenopus laevis frogs were obtained from Xenopus Express (Fort Atkinson,

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WI, USA), housed in an established frog colony and fed regular frog brittle on alternate days. The frog colony was maintained at Old Dominion University, Norfolk, Virginia, following the stipulations set by their Institute Animal Care and Use Committee (IACUC). The dissection of the frogs, removal of the oocytes and their defolliculation were conducted as described before [14-16].

Nuclear (germinal vesicle) injection was performed (Drummond “Nanoject,”

Drummond Scientific, Co., Broomall, PA, USA) using a maximum of 3 ng of cDNA containing a

ACCEPTED MANUSCRIPT 7 1:1:1 mix (molar ratio) of WT Cav2.3α1 or mutant Cav2.3α1, β1b and α2/δ cDNA subunits. The oocytes were incubated at 16°C for 7–8 days before the recording of current. 2.2 Construction of Mutants

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We used Cav2.3α1 subunit from the rat brain for the WT as well as the construction of the mutants. The selected Ser/Thr (Fig. 1) were mutated to Ala or Asp by primer extension using PCR with pfuTurbo DNA polymerase (QuickChange XL site-directed mutagenesis kit,

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Stratagene, La Jolla, CA, USA) as described before [4, 5]. The mutants were sequenced to confirm the mutation. Current recording

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2.3

Macroscopic currents with Ba2+ (IBa) as the charge carrier were recorded using a twoelectrode voltage clamp technique with oocyte Clamp OC-725C (Warner, Hamden, CT, USA) as described before [4, 5]. To construct the current-voltage (I–V) relationship for these currents,

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400 ms test potentials were employed starting from −50 to 100 mV with incremental step of 10 mV. The oocytes were held at -80 mV followed by pre-pulse voltage ranging from -95 to 40 mV with incremental steps of 5 mV for approximately 900 msec., before evoking a test pulse of 0 mV

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for a period of 100 msec., for recording the steady-state inactivation. PKC isozyme injection

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PKC isozymes (Sigma, St.Louis, MO, USA) were diluted to a stock solution of 20 nM

with frog ringer solution containing 30% (V/V) glycerol and stored at -80°C. Ten-fold dilution of the stock PKC isozymes (2 nM) was injected (27.6 nl) into each oocyte since these concentration gave significant responses. Considering an oocyte volume of approximately 1 µl and the dilution (27.6 nl of 2 nM PKC into 1 µl), the final concentration of the PKC isozyme injected was about 55 pM. Injection of PKC isozymes was carried out in the cold room (4° C)

ACCEPTED MANUSCRIPT 8 and subsequently the oocytes were transferred to the recording chamber; the IBa was recorded 2 min after the impalement.

Approximately a lag time of 15-30 min was required after the

injection of PKC isozymes and before testing their effect.

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2.5 Data Analysis

Results are expressed as means±SEM. Differences between groups were examined using Student’s t-test or one way ANOVA and a P-value of less than 0.05 was taken as significant. Results

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3.1 PKC isozymes βII- or ε-induced potentiation of Cav2.3 currents was alleviated by the quadruple mutation of selected Ser/Thr sites Both PKC isozymes increased the IBa induced during depolarization to 0 mV and I-V plot (Figs. 2A, B & C). I-V plot showed that the control IBa peaked at 10 mV and the same with PKC βII and ε at 0 and 10 mV respectively; isozyme-induced potentiation (p<0.01 to <0.05) of IBa was

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observed at several voltages in the I-V plot (Fig. 2C). Voltage-dependent inactivation of these channels in the control and in the presence of isozymes is nearly similar as the V1/2 for control, PKC βII and ε were -45.43, -48.59 and -48.06 mV respectively (Fig. 2D). However in the

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presence of PKCε, voltage-dependent inactivation of these channels deviated from the control at the depolarized potentials.

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The effects of PKC βII or ε were examined in the quadruple Ala mutation of the PMA sites, T365A/S369A/S1995A/S2011A (4A) [4] and both the PKC isozymes failed to affect the IBa when depolarized to 0 mV (Figs. 3A & B). I-V plot revealed that the IBa through the 4A mutant peaked at 10 mV similar to that of the WT and neither of these isozymes had any effect on the IBa (Fig. 3C). Voltage-dependence of 4A channel inactivation shifted (p<0.01 to 0.02) to more depolarized potentials and theV1/2 shifted towards the depolarizing direction (WT = -45.43 and 4A = -38.57 mV) as shown in Fig. 3D.

ACCEPTED MANUSCRIPT 9 3.2 Thr-365 and Ser-1995 mediate the stimulatory and Ser-369 the inhibitory functions of PKC isozymes Following the loss of PKC isozymes-induced potentiation of Cav2.3 currents in the 4A

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mutant, the response to PKC isozymes on individual Ser/Thr sites was examined. In this regard four triple Ala mutants were constructed in such a way that only one Ser/Thr was available in each construct. In the triple mutant Thr-365/S369A/S1995A/S2011A (T365/3A), IBa peaked at

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10 mV as that of the WT channels (Fig. 4B). Both the PKC isozymes increased the IBa in T365/3A as seen during depolarization to 0 mV (Fig. 4A) and the I-V plot (Fig. 4B). Voltage-

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dependent inactivation showed a biphasic pattern through the T365/3A channels. It shifted to hyperpolarizing voltages (p <0.01 to 0.05) when held at negative potentials and to depolarizing voltages (p <0.001 to 0.05) when held at positive potentials; furthermore, V1/2 shifted towards depolarizing direction by approximately 5 mV (Fig. 4C).

In the construct Ser-369/T365A/S1995A/S2011A (S369/3A), both PKC isozymes

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decreased the IBa (p<0.001) when depolarized to 0 mV (Fig. 5A). Although both the isozymes decreased the IBa (p<0.02 to <0.05) in the I-V plot, PKCβII appears to have more pronounced

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effect than PKCε (Fig. 5B). Voltage-dependent inactivation of S369/3A channels was almost similar to the WT, though V1/2 increased by 1 mV in the hyperpolarizing direction (Fig. 5C).

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Isozymes differed in their targeting of the triple mutant Ser-1995/T365A/S369A/S2011A (S1995/3A). Among the two isozymes, only PKC ε increased the IBa (p<0.001 to <0.05) when depolarized to 0 mV or I-V plot (Fig. 6 A & B). Voltage-dependent inactivation shifted to both hyperpolarized and depolarized potentials (p<0.001 to <0.05) and the V1/2 moved towards the hyperpolarizing direction (Fig. 6C). Both

PKCs

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mutant

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2011/T365A/S369A/S1995A (S2011/3A) as seen during depolarization to 0 mV and the I-V plot

ACCEPTED MANUSCRIPT 10 (Figs. 7 A & B). Voltage-dependent inactivation was peculiar through the S2011/3A channels. It shifted to hyperpolarizing voltages (p <0.001 to <0.05) when held at negative potentials and to

moved towards depolarizing direction (Fig. 7C). 3.3

The stimulatory and inhibitory effects neutralize each other

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depolarizing voltages (p <0.001 to <0.05) when held at positive potentials; furthermore, V1/2

Based on the data shown in Figs. 4 to 6, Thr-365 and Ser-1995 mediated the stimulatory

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action of PKC and Ser-369 the inhibitory action of PKC; Ser-2011 may not be a target of PKC isozymes. Hence we examined the relative dominance of Thr-365, Ser-369 and Ser-1995 with

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the constructs i) T365S369/S1995A and ii) S369S1995/T365A. In both the constructs neither PKC isozyme affected the IBa (Fig. 8 A & B). Subsequently we constructed another mutant in which both the stimulatory sites were available and the inhibitory site was mutated to Ala (T365S1995/S369A) in order to examine if there is any additional effect by the two stimulatory

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sites. Although both isozymes potentiated (p<0.02) the IBa through T365S1995/S369A compared to WT, there was no additional increase compared to Thr-365 (T365/3A) or Ser-1995 (S1995/3A) with either of the isozymes (Fig. 8C).

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3.4 Ser/Thr→ Asp mutation of the selected amino acids mimics the response of PKC isozymes In order to reproduce the effects of PKC isozymes on the selected Ser/Thr sites, these

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sites were mutated to aspartic acid (Asp) and the basal IBa was examined. As expected the basal IBa were increased with T365D and S1995D as well as in the double Asp mutant T365D/S1995D compared to WT; in contrast, basal IBa was decreased in S369D (Fig. 9 A). Both PKC isozymes increased the IBa through the WT (p<0.001) and S369D channels (p<0.01) (Figs. 9 B & C).In addition, PKCε decreased the IBa through T365D channels (p<0.01) as shown in Fig. 9C. 4.

Discussion

ACCEPTED MANUSCRIPT 11 Cav2.3 channels are a subject of PKC modulation - Cav2.3 channels are localized to somatic and dendritic membranes of the neurons [17-20] and may be responsible for dendritic excitability [21]. Some of these channels located at certain presynaptic terminals are reported to

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participate in evoked neurotransmitter release [22-24]; in addition, these channels may also be involved in hormonal secretion as they are expressed by various neuroendocrine cells [25-27]. Further these channels are demonstrated to be essential for certain forms of synaptic plasticity

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within mammalian hippocampus [28]. Knock-out of Cav2.3α1 subunit in mice led to functional deficits in fear behavior, spatial memory, pain perception and glucose metabolism [29-33]. PKC

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modulation, an observation made by several laboratories for Cav2.3 channels [5, 10-12] may be responsible for some of these functions based on the contribution of PKC in the function of nervous system.

It has been reported that Cav2.3α1 subunits may be the target of PKC

modulation of these channels and exposure to PKC activation delayed the inactivation of IBa

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expressed by Cav2.3α1 subunits alone. However, the presence of the auxiliary subunits, esp., the β subunits, further potentiated the PKC modulation of these channels [7, 34]. PKC isozymes have been reported to target the intracellular regions of Cav2.3α1 subunit,

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viz., I-II linker, II-III linker and C-terminus as shown by us and other laboratories [4, 5, 10, 13].

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Several Ser/Thr PKC target sites have been identified in the intracellular segments of Cav2.3α1 subunits and some of them are homologous (Fig. 1) to the Ser/Thr of Cav2.2α1 subunit, another member of Cav2 family that also undergoes modulation by PKC [4, 13, 15]. In spite of the homologous nature of these sites in their α1 subunits, the response to PKC isozymes differed between Cav2.2 and Cav2.3 channels. For example, in the presence of Ser-2011, PKCβII or ε failed to potentiate the IBa (see Figs. 7 A & B), contrary to its homolog, Ser-2132 in the Cav2.2α1 subunit [15]. Further, Ser-2132 was very potent as it increased the IBa even in the presence of the

ACCEPTED MANUSCRIPT 12 inhibitory site, Ser-425 [15]. Another C-terminal stimulatory site, Ser-1995, was targeted by PKCε only (see Figs. 6 A & B) although its homolog, Ser-2108 in the Cav2.2α1 subunit, was

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targeted by either PKC βII or ε [15]. It is possible that Ser-1995 is a weaker PKC site or it is a subject of selected PKC isozymes only. Being weaker may be the reason for Ser-1995 for not inducing additional potentiation on top of the increase caused by Thr-365 (see Figs. 6 and 8 C). It is likely that Thr-365 is a stronger stimulatory site as either PKC βII or ε potentiated the IBa when

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Thr-365 was alone (see Figs. 4 A & B). However the responses of Thr-365 and Ser-369 are in

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contrast to Thr-422 and Ser-425, their homologs in Cav2.2α1, as the effects through Thr-422 could not be neutralized by the inhibitory site, Ser-425 [15]. Though Thr-365 may be the stronger stimulatory site in the Cav2.3α1 subunit, it may be weaker than its homolog Thr-422 and hence neutralized by Ser-369. The other possibility is that Ser-369 is a potent inhibitor compared to its homolog, Ser-425. Support for the above statement arises from the observation that Ser-

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369 inhibited the responses of PKC βII and ε by 68% and 65% respectively (vs. 30% for PKCβII and 70% for PKCε by Ser-425) [15]. The Ser/Thr that mediates the inhibitory function of PKC

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may have additional importance as Ser-425 of Cav2.2α1 subunit has reduced Gβγ-mediated inhibition of Cav2.2 currents [13]. Such a role for Ser-369 is possible as inhibition of Cav2.3

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currents by Gβγ was blocked by PKC-dependent phosphorylation [11]. Furthermore, Ser-369 failed to contribute to steady state inactivation (Fig. 5C) contrary to all the stimulatory sites. Taken together, the differential responses of Ser/Thr sites of Cav2.2α1 and Cav2.3α1 subunits to PKC isozymes may be behind the functional variations of these channels. For example, while Cav2.3 channels were reported to contribute to insulin release [35, 36], the role of Cav2.2 channels in insulin secretion is doubtful [37, 38].

ACCEPTED MANUSCRIPT 13 Action of isozymes on stimulatory and inhibitory sites leads to physiological antagonism The stimulatory effects of PKCs mediated by Thr-365 or Ser-1995 were completely neutralized by Ser-369, the inhibitory site, when either Thr-365 or Ser-1995 were present together with Ser-

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369 (see Figs. 8A& B). Similarly PKCβII failed to alter the IBa in the Asp constructs T365D and S1995D, in which either of the stimulatory Ser-1995 or Thr-365 and inhibitory Ser-369 were available (see Fig. 9B). Taken together the lack of response in these constructs may be a case of

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physiological antagonism as PKC targeting of stimulatory and inhibitory sites neutralized each other. On the other hand, in spite of the presence of only the inhibitory Ser-369, there was no

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decrease of IBa through double Asp construct, T365D/S1995D by the PKC isozymes (see Figs. 9 B & C). This situation is due to the combined presence of two stimulatory sites, a condition that always dominated the inhibitory site, as seen in Cav2.3 WT. The domination of stimulatory sites over the inhibitory site is supported by the increased IBa with PKC βII or ε in S369D (see Figs. 9

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B & C). Taken together the inhibitory Ser-369 dominated the stimulatory sites when they were alone. In contrast, two stimulatory sites were required to neutralize and overcome the inhibitory Ser-369. Hence the net effect of PKC is the difference in the responses produced by Ser/Thr sites

5.

Conclusion

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that mediate the stimulatory and inhibitory actions of PKC isozymes.

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In summary, the differential responses seen with the PKC activators and modulators is likely due to the isozymes activated and the affinities of the target Ser/Thr PKC phosphorylation sites. The presence of basic and acidic amino acids in the vicinity of PKC targets may further affect the results with PKC isozymes. In addition the auxiliary Cavβ subunits may also selectively permit the action of PKC isozymes leading to the modulation of these channels. Taken together the effects of PKC isozymes do not depend on the target Ser/Thr PKC phosphorylation sites alone.

ACCEPTED MANUSCRIPT 14 Acknowledgments This work was supported by National Institutes of General Medical Sciences (SCORE3,

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GM096947) to G.L.K. We thank Dr. T. P. Snutch (University of British Columbia, Vancouver, British Columbia, Canada) for the clones of calcium channels. The laboratory of Dr. Logothetis, Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA 23298 is sincerely appreciated for helping with the oocyte digestion. Drs. Sacharia and Deo,

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Department of Engineering, Norfolk State University, Norfolk, VA 23504 are thanked for

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solving the electrical noise issue and helping with the data analysis.

ACCEPTED MANUSCRIPT 15 Conflict of Interest

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The authors declare no actual or potential conflict of interest.

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[33] S. Wilson, P. Toth, S. Oh, S. Gillard, S. Volsen, D. Ren, L. Philipson, E. Lee, C. Fletcher, L. Tessarollo, N. Copeland, N. Jenkins, R. Miller, J. Neurosci. 20 (2000) 8566-8571. [34] G. Kamatchi, S. Tiwari, C. Chan, D. Chen, S.-H. Do, M. Durieux, C. Lynch III, Brain. Res. 968 (2003) 227-237. [35] V. Schulla, E. Renstrom, R. Feil, S. Feil, I. Franklin, A. Gjinovci, X. Jing, D. Laux, I. Lundquist, M. Magnuson, S. Obermuller, C. Olofsson, A. Salehi, A. Wendt, N. Klugbauer, C. Wollheim, P. Rorsman, F. Hofmann, EMBO. J. 22 (2003) 3844-3854.

ACCEPTED MANUSCRIPT 18 [36] X. Jing, D.-Q. Li, C. Olofsson, A. Salehi, V. Surve, J. Caballero, R. Ivarsson, I. Lundquist, A. Pereverzev, T. Schneider, P. Rorsman, E. Renström, J. Clin. Invest. 115 (2005) 146-154. [37] A. Davalli, E. Biancardi, A. Pollo, C. Socci, A. Pontiroli, G. Pozza, F. Clementi, E. Sher, E. Carbone, J. Endocrinol. 150 (1996) 195–203.

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[38] E. Sher, E. Biancardi, A. Pollo, E. Carbone, G. Li, C.B. Wollheim, F. Clemnti, Euro. J.

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Pharmacol. 216 (1992) 407-414.

ACCEPTED MANUSCRIPT 19 Figure Legends Fig. 1 - Schematic diagram of Cav2.2α1 and Cav2.3α1 subunits showing the potential PKC

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phosphorylation sites. I-IV designate the transmembrane domains. The circles represent the amino acids and the numbers above them specify their positions in the Cavα1 subunits shown on the right; amino acids shown within the rectangles are homologous. ο, putative inhibitory PKC

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site, ●, putative stimulatory PKC site, T, threonine, S, serine and I-IV transmembrane domains.

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Fig. 2 - Effects of PKCs βII and ε on Cav2.3 currents expressed with Cav2.3α1β1bα2/δ subunits in Xenopus oocytes. Following the injection of the cDNA, the oocytes were incubated at 16°C for 7-8 days before the recording of the IBa. PKC isozymes were injected into the oocytes on the day of the experiment as mentioned in the methods. A and B top and middle panels, show the typical

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current tracings recorded at the test potential of 0 mV for a period of 900 msec.; the dashed lines indicate the zero current level; A and B bottom panels, illustrate the averaged peak IBa. C, illustrates the averaged I-V relationship for peak IBa recorded for a period of 400 msec. D, shows

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steady-state inactivation plotted for the Cav2.3 WT control (V1/2 = -45.43 mV), PKCβII (V1/2= 48.59 mV) and PKCε (V1/2= -48.06 mV) injected oocytes; the inset shows the schematics of

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steady state inactivation (prepotential and test potential are 900 and 100 msec., duration respectively) protocol. bp<0.01, cp<0.02 and dp<0.05 as compared with the control by one way ANOVA.‘n’ varied from 8 to 24 oocytes. The data points shown are mean ± SEM.

Fig. 3 - Effects of PKCs βII and ε on Cav2.3 quadruple mutant currents expressed with Cav2.3α1T365A/S369A/S1995A/S2011A (4A) with β1b and α2/δ subunits in Xenopus oocytes. A

ACCEPTED MANUSCRIPT 20 and B top and middle panels, show the typical current tracings recorded at the test potential of 0 mV for a period of 900 msec.; the dashed lines indicate the zero current level; A and B bottom panels, illustrate the averaged peak IBa. C, illustrates the averaged I-V relationship for peak IBa

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recorded for a period of 400 msec for the 4A mutant control and after isozyme treatment. D, compares the steady-state inactivation from the oocytes expressing Cav2.3 WT or 4A mutant channels; the prepotential and test potential (0 mV) were for a period of900 and 100 msec.

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respectively. bp<0.01, cp<0.02 and dp<0.05 as compared with the control by one way ANOVA.

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‘n’ varied from 8 to 21 oocytes. The data points shown are mean ± SEM.

Fig. 4 - Effects of PKCs βII and ε on Cav2.3 currents expressed byCav2.3α1subunit mutant Thr365/S369A/S1995A/S2011A (T365/3A) along with β1b and α2/δ subunits in Xenopus oocytes. Panel A shows the averaged peak IBa recorded at the test potential of 0 mV for a period of 900

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msec. B, illustrates the averaged I-V relationship for peak IBa recorded for a period of 400 msec. Panel C, shows the steady-state inactivation for Cav2.3WT and T365/3A; the prepotential and test potential (0 mV) were for a period of 900 and 100 msec. respectively. p<0.001,bp<0.01,cp<0.02and

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p<0.05 compared with the respective control by one way

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ANOVA. ‘n’ varied from 10 to 27 oocytes. The data points shown are mean ± SEM.

Fig. 5 - Effects of PKCs βII and ε on Cav2.3 currents expressed by Cav2.3α1 subunit mutant Ser369/T365A/S1995A/S2011A (S369/3A) along with β1b and α2/δ subunits in Xenopus oocytes. Panel A shows the averaged peak IBa recorded at the test potential of 0 mV for a period of 900 msec. B, illustrates the averaged I-V relationship for peak IBa recorded for a period of 400 msec. Panel C shows the steady-state inactivation for Cav2.3WT and S369/3A channels; the

ACCEPTED MANUSCRIPT 21 prepotential and test potential (0 mV) were for a period of 900 and 100 msec. respectively. a

p<0.001,cp<0.02and dp<0.05 compared with the respective control by one way ANOVA. ‘n’

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varied from 9 to 25 oocytes. The data points shown are mean ± SEM.

Fig. 6 - Effects of PKCs βII and εon Cav2.3 currents expressed by Cav2.3α1subunit mutant Ser1995/T365A/S369A/S2011A (S1995/3A) along with β1b and α2/δsubunits in Xenopus oocytes.

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Panel A shows the averaged peak IBa recorded at the test potential of 0 mV for a period of 900 msec. B, illustrates the averaged I-V relationship for peak IBa recorded for a period of 400 msec.

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Panel C shows the steady-state inactivation for Cav2.3WT and S1995/3A mutant channels; the prepotential and test potential (0 mV) were for a period of 900 and 100 msec. respectively. a

p<0.001,bp<0.01,cp<0.02and dp<0.05compared with the respective control by one way ANOVA.

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‘n’ varied from 8 to 36 oocytes. The data points shown are mean ± SEM.

Fig. 7 - Effects of PKCs βII and ε on Cav2.3 currents expressed by Cav2.3α1 subunit mutantSer2011/T365A/S369A/S1995A (S2011/3A) along with β1b and α2/δ subunits in Xenopus oocytes.

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Panel A shows the averaged peak IBa recorded at the test potential of 0 mV for a period of 900

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msec. B, illustrates the averaged I-V relationship for peak IBa recorded for a period of 400 msec. Panel C shows the steady-state inactivation for Cav2.3WT and S2011/3A mutant; the prepotential and test potential (0 mV) were for a period of 900 and 100 msec. respectively. a

p<0.001,cp<0.02and dp<0.05 compared with the respective control by one way ANOVA. ‘n’

varied from 7 to 23 oocytes. The data points shown are mean ± SEM.

ACCEPTED MANUSCRIPT 22 Fig. 8 - Effects of PKCs βII and ε on averaged peak IBa expressed by the single mutant of Cav2.3α1 subunits along with β1b and α2/δ subunits in Xenopus oocytes. The IBa was recorded at

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a test potential of 0 mV for a period of 900 msec. cp<0.02 as compared with the control by one way ANOVA. The numbers in parentheses indicate ‘n’. The data points shown are mean ± SEM.

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Fig. 9 - Effects of PKCs βII and ε on averaged peak IBa expressed by Cav2.3 WT orSer/Thr→

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Asp constructs of Cav2.3α1 along with β1b and α2/δ subunits in Xenopus oocytes. The IBa was recorded at a test potential of 0 mV for a period of 900 msec. ap<0.001 and bp<0.01 as compared with the Cav2.3 WT by Student’s t-test; *p<0.01 as compared with its own control by one way

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ANOVA. The numbers in parentheses indicate ‘n’. The data points shown are mean ± SEM.

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