Stimulation of recombinant Ca(v)3.2, T-type, Ca(2+) channel currents by CaMKIIgamma(C)

Abstract

Molecular cloning of low-voltage activated (LVA) T-type calcium channels has enabled the study of their regulation in heterologous expression systems. Here we investigate the regulation of Ca(v)3.2 alpha(1)-subunits (alpha1H) by calcium- and/or calmodulin-dependent protein kinase II (CaMKII). 293 cells stably expressing alpha1H were transiently transfected with CaMKIIgamma(C). Using the whole-cell recording configuration, we observed that elevation of pipette free Ca(2+) (1 microM) in the presence of CaM (2 microM) increases T-type channel activity selectively at negative potentials, evoking an 11 mV hyperpolarizing shift in the half-maximal potential (V(1/2)) for activation. The V(1/2) of channel inactivation is not altered by Ca(2+)/CaM. These effects reproduced modulation observed in adrenal zona glomerulosa cells. The potentiation by Ca(2+)/CaM was dependent on the co-expression of CaMKIIgamma(C) and required Ca(2+)/CaM-dependent kinase activity. Peptide (AIP) and lipophilic (KN-62) protein kinase inhibitors prevented the Ca(2+)/CaM-induced changes in channel gating without altering basal Ca(v)3.2 channel activity (27 nM free Ca(2+)) as did replacing pipette ATP with adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable analogue. CaMKII-dependent potentiation of channel opening resulted in significant increases in apparent steady-state open probability (P(o)) and sustained channel current at negative voltages. Under identical conditions, CaMKII activation did not regulate the activity of Ca(v)3.1 channels, the first cloned member (alpha1G) of the T-type Ca(2+) channel family. Our results provide the first evidence for the differential regulation of two members of the Ca(v)3 family by protein kinase activation and the first report reconstituting CaMKII-dependent regulation of any cloned Ca(2+) channel.

Figure 1. Elevation of pipette Ca²⁺/CaM fails to increase Ca²⁺ currents in 293 cells expressing Ca_v3.2 channels

Slowly deactivating tail currents were elicited at −60 mV after various depolarizing test pulses (V_t = −60 to +10 mV; 10 ms) from a holding potential of −90 mV. A, sample currents at V_t = −30 and +10 mV from two cells with intracellular free Ca²⁺ fixed at 27 nm Ca²⁺ (○) or 1 μm Ca²⁺ + 2 μm CaM (•). Note that the two cells have similar maximal currents (I_max). B, voltage dependence of activation. Normalized amplitude of slowly deactivating tail current (I/I_max) plotted (means ± s.e.m.) vs. V_t for two data sets. Data were fitted to a squared Boltzmann distribution yielding half-maximal activation potentials of: 27 nm Ca²⁺, V_1/2 = −23.7 ± 0.4 mV (k = 10.2, r = 0.99; n = 14 cells) and 1 μm Ca²⁺, V_1/2 = −26.8 ± 0.2 mV * (k = 10.2, r = 0.99, n = 9 cells, where k is the slope factor and r is the regression coefficient. *Not statistically different from 27 nm Ca²⁺ (by Student' unpaired t test).

Figure 2. Identification of two partial CaMKIIγ_C transcripts in bovine glomerulosa cells

Deduced amino acid sequences from two bovine glomerulosa cell RT-PCR products (γ_C and γ_X) amplified using bovine glomerulosa cell RNA and primers to highly conserved regions in the regulatory and association domains in all CaMKII gene families that spanned the intervening variable regions. The bovine sequences were aligned showing deletion regions (V1 and V2) absent in CaMKIIγ subunits previously designated as γ_C. The conserved domain separating the V1 and V2 regions was absent in γ_X. The sequences have been deposited in GenBank with the following accession numbers: bγ_C, AF 389986; and bγ_X, AF389987.

Figure 3. Elevation of pipette Ca²⁺/CaM induces a hyperpolarizing shift in the V_1/2 of activation of Ca_v3.2 channels in 293 cells expressing CaMKIIγ_C

Slowly deactivating tail currents were elicited at −60 mV, after various depolarizing test pulses (V_t = −60 to +10 mV; 10 ms) from a holding potential of −90 mV (activation) or after depolarizations (+20 mV, 8 ms) from various prepulse potentials (V_P = −90 to −20 mV) lasting 6 s (inactivation). A, activation. Sample currents at V_t = −55, −45, −35, −25, −5 or +5 mV from two cells with intracellular free Ca²⁺ fixed at 27 nm (○) or 1 μm + 2 μm CaM (•). Note the larger amplitude of currents at hyperpolarized potentials recorded with free Ca²⁺ fixed at 1 μm (•). B, voltage dependence of channel activation. Relative amplitude of tail currents (means ± s.e.m.) plotted vs. V_t for two data sets. Data were fitted to a squared Boltzmann distribution, yielding half-maximal activation potentials of: 27 nm Ca²⁺, V_1/2 = −23.1 ± 0.3 mV (k = 11.2, r = 0.99; n = 21 cells); and 1 μm Ca²⁺, V_1/2 = −34.0 ± 0.5 mV * (k = 10.2, r = 0.99, n = 21 cells). * Statistically different from 27 nm Ca²⁺ (P < 0.05 by Mann-Whitney unpaired non-parametric test). Inset, immunoblot of cell lysates from Ca_v3.2-expressing cells transfected with: CaMKIIγ_C and GFP (γ_C transfected), or GFP alone (untransfected). Recombinant CaMKIIγ_C protein served as standard. C, inactivation. Sample currents at V_P = −90, −70, −60, −55, −50 and −40 mV from two cells with intracellular free Ca²⁺ fixed at 27 nm (▵) or 1 μm + 2 μm CaM (▾). D, voltage dependence of channel inactivation. Relative amplitude of tail current (means ± s.e.m.) plotted vs. V_p for two data sets. Data were fitted to a Boltzmann distribution, yielding half-maximal inactivation potentials of: 27 nm Ca²⁺, V_1/2 = −67.1 ± 0.2 mV (k = 6.6, r = 0.99, n = 10 cells); and 1 μm Ca²⁺, V_1/2 = −67.2 ± 0.2 mV * (k = 6.8, r = 0.99, n = 9 cells). * Not statistically significant from 27 nm Ca²⁺ (by Student' unpaired t test).

Figure 4. Ca²⁺/CaM shifts the current-voltage relationship of Ca_v3.2 channels to negative potentials

A, mean traces (n = 10 and 11) at V_t = −50 mV show inward currents elicited from cells with pipette Ca²⁺ fixed at 27 nm (○) or 1 μm (•). B, I-V relationship. Peak inward current normalized to cell capacitance plotted vs. test potential for cells recorded with CaMKII-activating (n = 11 cells) or non-activating solutions (n = 10 cells). Note that with CaMKII-activating solutions Ca_v3.2 currents are enhanced within the range of −60 to −35 mV.

Figure 5. Autocamtide-2-related inhibitory protein (AIP), a specific peptide inhibitor of CaMKII, blocks Ca²⁺/CaM-induced potentiation of Ca_v3.2 currents in 293 cells expressing CaMKIIγ_C

A, representative traces at V_t = −30 mV and +10 mV show tail currents (V_r= −60 mV) elicited from two cells with 2 μm AIP in the pipette solution. Intracellular free Ca²⁺ was fixed at 27 nm (○) or 1 μm (•). Note that in the presence of AIP, Ca²⁺/CaM did not potentiate Ca_v3.2 currents. B, voltage dependence. Relative amplitude of tail current (means ± s.e.m.) plotted vs. V_t for two data sets. Half-maximal activation potentials were calculated as in Fig. 3B; 27 nm Ca²⁺ + AIP, V_1/2 = −21.5 ± 0.3 mV (k = 12.4, r = 0.99, n = 12 cells); and 1 μm Ca²⁺ + AIP, V_1/2 = −22.7 ± 0.5 mV * (k = 12.4, r = 0.99, n = 11 cells). * Not statistically significant from 27 nm Ca²⁺ + AIP (by Student' unpaired t test). Dashed lines show comparison fits to data points obtained at 27 nm and 1 μm Ca²⁺ without AIP in the pipette, data from Fig. 3B.

Figure 6. CaMKII activity is required for Ca²⁺/CaM to modify the gating of Ca_v3.2 channels

V_1/2 of activation (means ± s.e.m., n = number of cells) was calculated for each cell recorded with 1 μm or 27 nm Ca²⁺ in the absence and presence of kinase inhibitors in the bath or pipette solutions. Dashed line indicates baseline V_1/2 determined in Fig. 3B at 27 nm Ca²⁺ in the absence of inhibitors. * Statistically significant from 27 nm Ca²⁺ (P < 0.05 by non-parametric one-way ANOVA). AMP-PNP (3 μm), poorly hydrolysable ATP analogue; AIP (2 μm), specific CaMKII-peptide inhibitor; and KN-62 (3 μm), membrane-permeable CaMKII-inhibitor, significantly reduced Ca²⁺-induced gating change. KN-04 (3 μm), inactive CaMKII-inhibitor, preserved gating change. Neither AIP nor KN-04 (not shown) changed control V_1/2 of activation at 27 nm Ca²⁺.

Figure 7. CaMKII activation increases Ca_v3.2 window current

A, predicted voltage range for observable Ca_v3.2 window currents. Overlap of Boltzmann distributions describing the voltage dependence of Ca_v3.2 activation (m_∞²) and inactivation (h_∞) determined from data in Fig. 3 defines voltage ranges for discernible window currents. Data were obtained with non-activating (continuous lines) or CaMKII-activating (dashed lines) pipette solutions. Note, the more hyperpolarized voltage range defined for CaMKII-activated cells. B, estimated steady-state channel open probability (P_o). Theoretical steady-state P_o (m_∞²h_∞) for Ca_v3.2 channels plotted vs. membrane potential for control (continuous line) and CaMKII-activated (dashed line) cells. Note, at −55 mV CaMKII activity increases P_o 295 %. C, predicted steady-state window current. Theoretical steady-state current calculated as: I = ((m_∞²h_∞)g N (V_t - V_r)), assuming g = 4.7 pS in 10 mm Ca²⁺, N = 13 400 Ca_v3.2 channels per cell, and that V_reversal = +52 mV. Note that CaMKII activity is predicted to increase Ca_v3.2 steady-state current by 295 % at −55 mV and 220 % at −80 mV. D, Ca²⁺ channel currents. Representative traces averaged from two cells at V_t = −60, −55, −50, −45, −35 and −30 mV. Intracellular free Ca²⁺ was fixed at 27 nm (upper panel) or 1 μm (lower panel). Note sustained Ca²⁺ channel current at 600 ms. Scale bar applies to both plots. E, measured channel open probability (P_o). Slowly deactivating tail currents (V_r = −90 mV) elicited following a 600 ms depolarization (V_t as in D) were averaged among cells with intracellular free Ca²⁺ fixed at 27 nm (○, n = 7 cells) or 1 μm (•, n = 8 cells). Relative amplitude of averaged tail current plotted vs. V_t for two data sets. The tail I_max (determined at V_r = −90 mV following V_t to +20 mV for 10 ms) at 27 nm = 3423 pA (n = 7 cells) and at 1 μm = 2897 pA (n = 8 cells). Note that at −55 mV CaMKII activity increases P_o by 195 %. F, maintained Ca²⁺ channel currents. Representative traces averaged from two cells at V_t = −50 and −45 mV where channel open probability is greatest. Intracellular free Ca²⁺ was fixed at 27 nm (upper panel) or 1 μm (lower panel). Scale bar applies to both plots. Both inward current and tail current amplitudes are increased by CaMKII.

Figure 8. Elevation of pipette Ca²⁺/CaM fails to increase Ca_v3.1 currents

A, representative traces at V_t −30 mV, +10 mV in two cells with intracellular Ca²⁺ fixed at 27 nm (○) or 1 μm (•). Note Ca²⁺/CaM did not potentiate Ca_v3.1 currents. B, voltage dependence. Relative amplitude of tail current (means ± s.e.m.) plotted vs. V_t for two data sets. Half-maximal potentials were calculated as above; 27 nm Ca²⁺, V_1/2 = −30.1 ± 0.3 mV (k = 8.2, r = 0.99, n = 12 cells); and 1 μm Ca²⁺, V_1/2 = −32.1 ± 0.3 mV * (k = 7.9, r = 0.99, n = 10 cells). * Not statistically different (by Student' unpaired t test). Inset, immunoblot of cell lysates from Ca_v3.1-expressing cells transfected with: CaMKIIγ_C and GFP (γ_C transfected), or GFP alone (untransfected). Recombinant CaMKIIγ_C protein served as standard. Note inadequate expression of CaMKIIγ_C cannot account for the lack of Ca_v3.1 regulation by CaMKIIγ_C.