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The HOOK region of β subunits controls gating of voltage-gated Ca2+ channels by electrostatically interacting with plasma membrane

ABSTRACT Recently, we showed that the HOOK region of the β2 subunit electrostatically interacts with the plasma membrane and regulates the current inactivation and phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity of voltage-gated Ca2+ (CaV) 2.2 channels. Here, we report that voltage-dependent gating and current density of the CaV2.2 channels are also regulated by the HOOK region of the β2 subunit. The HOOK region can be divided into 3 domains: S (polyserine), A (polyacidic), and B (polybasic). We found that the A domain shifted the voltage-dependent inactivation and activation of CaV2.2 channels to more hyperpolarized and depolarized voltages, respectively, whereas the B domain evoked these responses in the opposite directions. In addition, the A domain decreased the current density of the CaV2.2 channels, while the B domain increased it. Together, our data demonstrate that the flexible HOOK region of the β2 subunit plays an important role in determining the overall CaV channel gating properties.


Introduction
Voltage-gated Ca 2C (Ca V ) channels adjust the Ca 2C influx in excitable cells. The channels contribute to the diverse physiologic responses, including neurotransmission, hormone secretion, muscle contraction, and gene transcription. [1][2][3] Dysregulation of Ca V channels causes various neurologic disorders, such as autism, migraine, and pain. [4][5][6][7] A high-voltage activated (HVA) Ca V channel consists of the pore-forming a1 subunit and the auxiliary b and a2d subunits. Surface expression of the a1 subunit requires the auxiliary subunits, where they also play critical roles in modulating the biophysical properties of Ca V a1 channel gating. [8][9][10] Among the auxiliary subunits, the b subunit is particularly important in regulating the gating and membrane expression of Ca V channels. [11][12][13] The b subunit can be divided broadly into 5 regions, such as the N and C terminus, the Src homology 3 (SH3), guanylate kinase (GK) domains, and the flexible HOOK region connecting the 2 domains. [12][13][14][15][16][17][18] Among the 5 regions of b subunit, N-terminus plays a key role in determining the subcellular localization of the b subunit, which is principally engaged in modulating the gating kinetics and membrane phospholipid sensitivity of Ca V channels. [19][20][21][22][23] The Ca V channel with membrane-anchored b subunit, such as b2a or b2e, exhibits relatively slow current inactivation and low sensitivity to the depletion of phosphatidylinositol 4,5-bisphosphate (PIP 2 ), whereas channels with cytosolic b subunit, such as b2b or b3, exhibit the opposite responses.
In our recent works, we found that the HOOK region of the b subunit acts as an important regulator for inactivation kinetics and membrane PIP 2 sensitivity of the Ca V 2 channels via dynamic electrostatic interaction of the b subunit with the plasma membrane. 24 The HOOK region can be further divided into 3 domains on the basis of amino acid composition: S (polyserine), A (polyacidic), and B (polybasic). Acidic residues within the A domain are needed for the increase of channel inhibition by PIP 2 depletion and the fast inactivation of Ca V 2.2 current like cytosolic b subunits, while basic amino acids within the B region are needed for the decrease in PIP 2 sensitivity and the slow inactivation like membrane-localized b subunits. Therefore, the regulatory mechanism of the Ca V 2 channels by the HOOK region looks very similar to the gating regulation by N-terminus-dependent subcellular localization of the b subunit.
It has been known that the subcellular localization of the b subunit is important for determining the current density of the Ca V channels. 19 The Ca V 2.2 channels with the cytosolic b3 subunit show significantly lower current density than channels with the membrane-tethered b2a subunit. The Lyn-b3 subunit which is located at the plasma membrane by adding the membrane-targeting Lyn sequence to the cytosolic b3 increases the current density of the Ca V 2.2 channels, whereas mutant b2a(C3,4S) which localizes in the cytosol by disabling the 2 N-terminal palmitoylation sites in the b2a subunit decreases the current density of the channels. This suggests that the gating properties of the Ca V channels including the inactivation kinetics, PIP 2 sensitivity, and current density, are commonly regulated by N-terminusdependent membrane interaction of the b subunit with the plasma membrane. Here, we further investigated the functional roles of the HOOK region of the b subunit on the gating of Ca V 2.2 and Ca V 1.3 channels. Our data demonstrate that through the interaction with the plasma membrane, the HOOK region of the b2c subunit also determines both voltagedependent gating properties and the current density of the Ca V channels.

Results
The HOOK region of the b2c subunit affects the voltage-dependent gating of the Ca V 2.

channels
We recently showed that the HOOK region of the b2 subunit is crucial for determining the inactivation kinetics and PIP 2 sensitivity of the Ca V 2.2 channels through dynamic interaction with the plasma membrane. 24 Here, we examined whether the HOOK region of the b subunit also affects the voltage-dependent inactivation (VDI) and voltage-dependent activation (VDA) of the Ca V channels. Fig. 1A shows the deletion mutants of the b2c HOOK region. When the b2c derivatives were expressed with N-type Ca V 2.2 channels, the VDI of the Ca V 2.2 channels with b2c(B) shifted to more positive voltages compared with the b2c control, whereas those channels with b2c(A) shifted to more negative voltages ( Fig. 1B and D Left).
In contrast, the VDA of the Ca V 2.2 channels with b2c (B) shifted to more negative voltages compared with the b2c control, whereas those channels with b2c(A) shifted to more positive voltages ( Fig. 1C and D Right). Channels with b2c(S) and b2cDHOOK did not show any significant changes in VDI and VDA ( Fig. 1B-D). Those results suggest that the charged amino acids in the HOOK region plays a key role in determining the voltage-dependent gating properties of the Ca V 2.2 channels.
Charged amino acids in the HOOK region of b subunit determines the current density of the Ca V 2.2 channels Because the subcellular localization of the Ca V b subunit is important in determining the current density of the Ca V channels, 19 we examined whether the HOOK region of the b2c subunit also affects the current density of the Ca V 2.2 channels. Fig. 2 shows that the relationship between voltage and current density of the Ca V 2.2 channels with b2c(S) or b2cDHOOK was almost the same as those of channels with the b2c control. However, the Ca V 2.2 channels with b2c(A) showed dramatically lower current density, whereas the channels with b2c(B) showed relatively higher density ( Fig. 2A and C).
Since charged amino acids of the HOOK region are mainly important in regulating inactivation kinetics and PIP 2 sensitivity of Ca V 2.2 channels, 24 we examined whether the charged amino acids of the HOOK region also influence the current density of the Ca V 2.2 channels. As shown in Fig. 3A and B, . Full length of b2c consists of the N-and C-terminus, the highly conserved SH3 and GK domain (gray) and HOOK region (green, blue and red). The HOOK region of b2c was separated into 3 domains, S (poly-serine), A (poly-acidic) and B (poly-basic). Serine residues within S-domain are marked in green, acidic residues within A-domain are in blue, and basic residues within B-domain are in red, respectively. (B) The pulse protocol was composed of a 5-s prepulse from -110 mV to 30 mV in 10 mV steps, followed by a 40-ms test pulse to 10 mV. Holding potential is -80 mV (Left, Top). Representative Ba 2C current traces were elicited by the pulse protocol (Left, Bottom). Peak point of test pulse currents following 5-s prepulse were normalized to the largest current in each series of test pulse. The curves were fitted by a Boltzmann equation. The voltage dependence of normalized steady-state inactivation for Ca V 2.2channels with b2c mutant derivatives (purple lines, Right). Data from control b2c (gray trace) are reproduced to facilitate visual comparison. (C) The pulse protocol was composed of a 40-ms test pulse from -70 mV to 70 mV in 10 mV steps. The membrane potential was held at -80 mV (Left, Top). Representative Ba 2C current traces were elicited by the pulse protocol (Left, Bottom). Tail currents following test pulses were normalized to the largest tail current in each series of test pulse. The curves were fitted by a Boltzmann function. The voltage dependence of normalized steady-state activation for Ca V 2.2channels with b2c mutant derivatives (orange lines, Right). Data from control b2c (gray trace) are reproduced to facilitate visual comparison. (D) The magnitude of changes in the V1/2 of normalized steady-state inactivation (purple bars, left) and activation (orange bars, right) from the mean of b2c control in cells expressing Ca V 2.2channels with b2c mutant derivatives. Dot lines indicate the b2c control level. Dots display the individual data points for each experiment. ÃÃÃ P < 0.001, one-way ANOVA followed by Dunnett's post-hoc test. Data are mean § SEM. channels with complete or partial deletion of the A domain and charge-neutralizing mutations in A domain of the b2c subunit showed significantly higher current density than the b2c control, whereas channels with complete deletion of the B domain and charge-neutralizing mutations in B domain showed lower current density ( Fig. 3C and D). These results suggest that acidic amino acids of the A domain play a role for decreasing the current density of the Ca V 2.2 channels, whereas basic amino acids of the B domain are important for increasing the response. We recently found that 2 hydrophobic Phe residues in the B domain are needed for decreasing the inactivation kinetics and PIP 2 sensitivity of the Ca V 2.2 channels. 24 Consistently, the Phe-mutated form (b2cPhe Ala ) also slightly decrease the current density of the channels (Fig. 3C and D). Together with the findings of our recent study, the present results indicate that dynamic interaction between the HOOK region and the plasma membrane through charged amino acids in the A and B domains are crucial for determining the current density of the Ca V 2.2 channels.
The HOOK region of the b subunit modulates the PIP 2 sensitivity and inactivation kinetics of the Ca V 1.3 channels We reported that N-type Ca V 2.2 channel gating is tightly modulated by the electrical properties of the HOOK region in the b2c subunit. 24 We then examined whether the HOOK region also influences the PIP 2 sensitivity and inactivation kinetics of L-type Ca V 1.3 channels. As shown in Fig. 4A, B, and C, Ca V 1.3 channels with b2c(A) show faster current inactivation, whereas channels with b2c(B) show slower inactivation. The fast inactivation kinetics of the Ca V 1.3 channels with b2c(S) and b2cDHOOK were slightly decreased but not significantly different compared with the Ca V 1.3 channels with the b2c control. The Ca V 1.3 channels with b2c(A) showed higher sensitivity to PIP 2 depletion mediated by the activation of zebrafish form of voltage-sensing phosphatase (Dr-VSP), whereas channels with b2c(B) showed lower inhibition to PIP 2 depletion (Fig. 4D and E). These results demonstrate that the current inactivation

Discussion
We recently found that the HOOK region of the b2 subunit regulates the inactivation kinetics and PIP 2 sensitivity of Ca V 2.2 channels via the electrostatic interaction with phospholipids in the plasma membrane. Here, our results enlarge our understanding for functional effects of the HOOK region on Ca V channel regulation. (a) The HOOK region of the b2c subunit regulates the voltage-dependent gating of Ca V 2.2 channels. The b2c subunit containing only the A domain in the HOOK region shifted the VDI and VDA to more negative and positive voltages, respectively, whereas the b2c subunit with only the B domain triggered the responses in the opposite directions. (b) The charged amino acids of the A and B domains are also crucial in determining the current density of the Ca V 2.2 channels. Acidic residues within the A domain plays an important role in decreasing the current density of the Ca V 2.2 channels, whereas basic residues within the B domain are important in increasing it. (c) The inactivation kinetics and PIP 2 sensitivity of the Ca V 1.3 channels are also regulated by the HOOK region of the b2c subunit. Together, our results suggest that the HOOK region determines the channel gating and current density of HVA a1 types.
It has been reported that subcellular localization of Ca V b subunits is important in regulating the biophysics and PIP 2 sensitivity of the Ca V channels. 19 Our recent data showed that the net surface charge of dynamic HOOK region of b subunits also performs similar functions in regulating the Ca V channel gating. 24 We found that the net charge of HOOK region is mainly determined by the exposure of either A-or B-domain to the b-subunit surface. In resting state, Aand B-domains of HOOK region seem to be masking each other through electrostatic interaction, making the HOOK region be more neutral. When the net charge of HOOK region is changed to the basic by exposing B-domain, the b subunit can further move to the plasma membrane and electrostatically interact with phospholipids via the basic HOOK region. When the net charge of HOOK region, meanwhile, is changed to acidic due to A-domain exposure, the b subunit will move toward the cytosol by repulsion between acidic phospholipids in the plasma membrane and the acidic HOOK region. We found that membrane-interacting b subunit of the Ca V channels through either N-terminus or HOOK region commonly slows inactivation, enhances PIP 2 sensitivity, and increases the current density of the Ca V channels. The reason why the Ca V channels with membraneinteracting b subunit exhibit higher current density than the channels with cytosolic b subunit remains unclear. There may be 2 possibilities. First, the membrane-interacting b subunit enhances the expression level of the Ca V channel complex in the plasma membrane. Recently, it was reported that the other auxiliary a2d subunit elevates the current density by enhancing the channel trafficking to the plasma membrane. 8 Second, the membrane-tethering b subunit alters the kinetics of channel gating and thus increases the open time of the channel gate. However, in the presence of a1B and a2d1, the diverse b2c-deletion mutants are present in the plasma membrane, 24 suggesting that the HOOK region of the b2c subunit does not influence the formation of heteromeric Ca V channel complex in the plasma membrane. Further studies are needed to define which is important in the gating control of the Ca V channels by the membrane-interacting b subunit. It is also reported that N-terminusdependent subcellular localization of the b subunit regulates the gating of L-type Ca V 1.3 and P/Q-type Ca V 2.1 channels. 19 Similarly, the phenomena also appeared in our experiments using the mutants of the HOOK region in the Ca V 1.3 channels, suggesting that the regulation of the Ca V channel gating by the N-terminus or HOOK region of the b subunit seems to be a general mechanism for all types of HVA a1 subunit.
In conclusion, our findings provide another modulatory mechanism of Ca V channel gating through the dynamic interaction of the HOOK region of the b subunit with the plasma membrane. Recently, it has been reported that the Ca V channel gating can be regulated by induced anchoring of intracellular loops of channels to the plasma membrane. 26,27 Those studies suggested a possibility that by interacting with the b subunit, the intracellular I-II loop of the Ca V channel plays a significant role in regulating the Ca V channel gating. Further studies are needed to investigate whether the conformational change of the I-II loop using the interacting membrane via the b subunit determines the Ca V channel gating in cells.

Data analysis
Pulse/Pulse Fit 8.11 software and an EPC-10 patch clamp amplifier (HEKA) were used for data acquisition and analysis. Further data processing used Microsoft Excel, WaveMetrics Igor Pro, and GraphPad Prism version 5.01, as described previously. 24 Voltage dependence of steady-state activation and inactivation was fitted by the Boltzmann function of the form 1/(1Cexp[-(V-V 1/2 )/k]), where V 1/2 is the half-maximal voltage for activation or inactivation and K is a slope factor. The time course of current inactivation was fitted by the double exponential function of the form as described previously. 24 All quantitative data were presented as the mean § SEM. Statistical significance was analyzed using one-way ANOVA, followed by Dunnett's post-hoc test ( Ã P < 0.05, ÃÃ P < 0.01, and ÃÃÃ P < 0.001).

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.