Cytoskeletal disrupting agents prevent calmodulin kinase, IQ domain and voltage-dependent facilitation of L-type Ca2+ channels

Abstract

A calmodulin (CaM) binding 'IQ' domain on the L-type Ca(2+) channel (LTCC) C terminus and calmodulin kinase II (CaMK) both signal increases in LTCC opening probability (P(o)) by shifting LTCCs into a gating mode (mode 2) with long openings through a process called facilitation. However, the mechanism whereby CaMK and the IQ domain are targeted to LTCCs is unknown. Endogenous CaMK is targeted to LTCCs in excised cell membrane patches because LTCC P(o) increased significantly in CaM-enriched (20 microM) bath solution and this effect was prevented by a specific CaMK inhibitory peptide, but not by an inactive control peptide. Pre-exposure of myocytes to the cytoskeletal disrupting agents nocodazole (microtubule specific) or cytochalasin D (microfilament specific) prevented the effects of CaM-dependent increases in P(o) of LTCCs in excised membrane patches. Neither cytochalasin D nor nocodazole altered the distribution of LTCC gating modes under basal conditions in on-cell mode or excised cell membrane patches, but each of these agents occluded the response of LTCCs to exogenous, constitutively active CaMK and to an IQ-mimetic peptide (IQmp). Cytochalasin D and nocodazole pretreatment also prevented LTCC facilitation that followed a cell membrane depolarizing prepulse. In contrast, cytochalasin D and nocodazole did not affect the increase in LTCC P(o) or prevent the shift to mode 2 gating in response to protein kinase A, indicating that cytoskeletal disruption specifically prevents prepulse, CaMK and IQ-dependent LTCC facilitation.

Figure 1. Cytoskeletal proteins are required for endogenous CaMK to facilitate L-type Ca²⁺ channels (LTCC) in excised cell membrane patches

A-C show LTCC opening probability (P_o) diary plots for 500 depolarizing steps. Exposure to high CaM (20 μm) solution increases channel activity (B) compared with control conditions (5 μm CaM, A). C, the effect of increased CaM was blocked by adding AC3-I (10 μm), a specific CaMK inhibitory peptide. D, summary data for LTCC P_o under control conditions (n = 6), 20 μm CaM (n = 5), 20 μm CaM with an inactive control peptide, AC3-C (10 μm, n = 4), 20 μm CaM and AC3-I (n = 4), and 20 μm CaM after treatment with cytochalasin D (Cyto-D, n = 7) or nocodazole (Noco, n = 8). *P < 0.05 compared with control.

Figure 2. The effect of cytoskeletal disruption on CaMK-, IQmp- and PKA-mediated increases in LTCC P_o

A-C show single channel records following exposure to exogenous, constitutively active CaMK (top) with superimposed ensemble-averaged currents before and after CaMK application. The effect of CaMK (indicated by the horizontal bar) on P_o during an ensemble of 500 depolarizing steps is also shown (bottom). DMSO control, cytochalasin D and nocodazole treatment conditions are indicated above each panel. D-F are ordered as in A-C, but show single LTCC records following exposure to IQmp (10 μm), indicated by the horizontal bar. G-I are ordered as the previous panels, but show the effect of the catalytic subunit of PKA (1 μm, top) on P_o, indicated by the horizontal bar.

Figure 3. Cytochalasin D (Cyto-D) and nocodazole (Noco) prevent increased LTCC P_o by CaMK and IQmp, but leave PKA signalling intact

Summary data for LTCC P_o from on cell mode (A) and excised cell membrane patch recordings (B) following treatment with DMSO (control), cytochalasin D or nocodazole. Neither cytochalasin or nocodazole affect LTCC P_o compared with control. However, both constitutively active CaMK (C) and IQmp (D) fail to increase LTCC P_o after treatment with cytochalasin D or nocodazole. E, in contrast, PKA-induced increases in LTCC P_o are not affected by cytochalasin D or nocodazole compared to control. Numerals (abscissa) indicate the number of LTCCs studied in each condition. *P < 0.05 compared with control.

Figure 4. Cytochalasin D and nocodazole prevent LTCC facilitation by CaMK and IQmp, while PKA-mediated LTCC facilitation is preserved

Summary data for the distribution of LTCC gating modes from on-cell mode (A) and excised cell membrane patch recordings (B) following treatment with cytochalasin D, nocodazole or DMSO (control). Neither cytochalasin D or nocodazole affect the distribution of gating modes compared with control. However, both constitutively active CaMK (C) and IQmp (D) fail to increase the proportion of sweeps with prolonged channel openings (mode 2) after treatment with cytochalasin D or nocodazole, compared to control. E, in contrast, cytochalasin D or nocodazole do not affect PKA-mediated increases in mode 2 gating sweeps. *P < 0.05 compared with cytochalasin D and nocodazole treatment. Analysis of LTCC gating modes (Yue et al. 1990) for cells treated with CaMK (F), IQmp (G) and PKA (H) after treatment with cytochalasin D. Only PKA was capable of increasing the probability of LTCCs entering into a gating mode with prolonged openings (mode 2) after treatment with cytoskeletal disrupting agents. Numerals indicate gating modes 0, 1 and 2 (see Methods for details). All analyses were performed on n = 3 LTCCs.

Figure 5. Cytoskeletal disrupting agents prevent prepulse (PP) LTCC facilitation

A, schematic representation of the voltage clamp protocol for eliciting PP facilitation. Control measurements omitted the PP and had a constant holding potential of −80 mV (dashed line). LTCC P_o was measured during the depolarizing step to 0 mV. B, mean LTCC P_o for cells treated with DMSO control vehicle, cytochalasin D (Cyto D) and nocodazole (noco). The number of LTCCs studied is indicated on the abscissa. *P + 0.003 compared with other groups.