Vascular Kv7 channels control intracellular Ca2+ dynamics in smooth muscle

Abstract

Voltage-gated Kv7 (or KCNQ) channels control activity of excitable cells, including vascular smooth muscle cells (VSMCs), by setting their resting membrane potential and controlling other excitability parameters. Excitation-contraction coupling in muscle cells is mediated by Ca²⁺ but until now, the exact role of Kv7 channels in cytosolic Ca²⁺ dynamics in VSMCs has not been fully elucidated. We utilised microfluorimetry to investigate the impact of Kv7 channel activity on intracellular Ca²⁺ levels and electrical activity of rat A7r5 VSMCs and primary human internal mammary artery (IMA) SMCs. Both, direct (XE991) and G protein coupled receptor mediated (vasopressin, AVP) Kv7 channel inhibition induced robust Ca²⁺ oscillations, which were significantly reduced in the presence of Kv7 channel activator, retigabine, L-type Ca²⁺ channel inhibitor, nifedipine, or T-type Ca²⁺ channel inhibitor, NNC 55-0396, in A7r5 cells. Membrane potential measured using FluoVolt exhibited a slow depolarisation followed by a burst of sharp spikes in response to XE991; spikes were temporally correlated with Ca²⁺ oscillations. Phospholipase C inhibitor (edelfosine) reduced AVP-induced, but not XE991-induced Ca²⁺ oscillations. AVP and XE991 induced a large increase of [Ca²⁺]_i in human IMA, which was also attenuated with retigabine, nifedipine and NNC 55-0396. RT-PCR, immunohistochemistry and electrophysiology suggested that Kv7.5 was the predominant Kv7 subunit in both rat and human arterial SMCs; CACNA1C (Cav1.2; L-type) and CACNA1 G (Cav3.1; T-type) were the most abundant voltage-gated Ca²⁺ channel gene transcripts in both types of VSMCs. This study establishes Kv7 channels as key regulators of Ca²⁺ signalling in VSMCs with Kv7.5 playing a dominant role.

Keywords: Calcium; Kv7; Phospholipase C; Retigabine; T-type Ca(2+)channels; Vascular smooth muscle cell; Vasopressin.

Graphical abstract

Fig. 1

Contribution of L-and T-type VGCCs to depolarisation-induced Ca²⁺ transients in A7r5 cells. (A) Representative example trace showing rises in [Ca²⁺]_i (indicated as F340/F380 ratio units; r.u.) evoked by depolarising cells with 50 mM K⁺-containing buffer (the period indicated by the solid bar). (B) Example traces of high-K⁺-induced Ca²⁺ transients recorded in the presence of L-type (nifedipine; 2 μM) or T-type (NNC 55-0396; 3 μM) Ca²⁺ channel blockers (as indicated). (C) Bar graph showing the mean area under the curve of the response to 50 mM K⁺ buffer (control group represented in panel A) and cell groups in the presence of nifedipine or NNC 55-0396 (represented in panel B). (D) Agarose gels stained with SYBR safe to visualise the RT-PCR products corresponding to L-type (Cacna1s, Cacna1c, Cacna1d, Cacna1f) and T-type (Cacna1g, Cacna1h, Cacna1i) VGCCs genes. (E) Quantification of RT-PCR results exemplified in panel D; expression is normalised to that of a housekeeping gene, Hprt1 (hypoxanthine phosphoribosyltransferase 1). In panels C and E data are presented as mean ± S.E.M.; *P < 0.05, **P < 0.01, ****P < 0.0001 (panel C, n = 5; panel E, n≥4).

Fig. 2

Kv7 channel inhibition induces Ca²⁺ oscillations linked to L- and T-type VGCCs activity. (A) Representative example trace showing Ca²⁺ oscillations (indicated as F340/F380 ratio units; r.u.) evoked by Kv7 channel inhibitor XE991 (10 μM). (B) Comparison of amplitude of Ca²⁺ signals induced by XE991 and 50 mM K⁺, estimated as area under the curve during first 50 s of stimulus application (corresponding to the duration of high-K⁺ stimulation; exemplified in the inset on the right). (C-E) Example traces of XE991-induced Ca²⁺ transients recorded in the presence of Kv7 channel opener, retigabine (10 μM; C), L-type (nifedipine; 2 μM; D) or T-type (NNC 55-0396; 3 μM; E) Ca²⁺ channel blockers (as indicated). (F) Upper panel: superimposed are Fura2 ratiometric Ca²⁺ recording (black) and FuoVolt membrane potential recording (measured as ΔF/F₀; red) during application of XE991 (10 μM) and retigabine (10 μM) during periods indicated by horizontal bars. Lower panel: Cross correlation of the normalised ratiometric Ca²⁺ signal with the normalised FluoVolt signal indicated the time lag between the signals at which the peak correlation occurred. (G-I) Bar graphs summarising the effects of retigabine (G), nifedipine (H) or NNC 55-0396 (I) on the XE991-induced Ca²⁺ spike frequency (spikes/s). Control is the spike frequency in the presence of XE991 measured from the onset of the first spike. For quantification of drug effect spike frequency was calculated from the onset of the drug application and until the end of the application of XE991. In panels G-I data are presented as mean ± S.E.M.; **P < 0.01, ***P < 0.001 (n = 5).

Fig. 3

Expression of Kcnq genes and Kv7 proteins in A7r5 cells. (A) Agarose gels stained with SYBR safe to visualise the RT-PCR products of Kcnq1, Kcnq2, Kcnq3, Kcnq4 and Kcnq5. (B) Quantification of RT-PCR results exemplified in panel A, expression is normalised to that of a housekeeping gene, Hprt1 (hypoxanthine phosphoribosyltransferase 1). (C) Immunofluorescence labelling of Kv7.1 – Kv7.5 channel subunits in A7r5 cells, scale bars are 20 μm. In panel B data are presented as mean ± S.E.M.; ***P < 0.001 (n = 6).

Fig. 4

XE991 elicits a partial blockade of current through Kv7.5/Kv7.3 heteromeric channels that can be fully recovered by retigabine. (A) Current voltage relationship of Kv7.4 (n = 8) and Kv7.5/3 (n = 10) channels prior to and post XE991 (10 μM) treatment. (B,C) Representative voltage clamp recordings at −20 mV showing effects of XE991 (10 μM) and retigabine (10 μM; applied in the presence of XE991) on Kv7.4 (B) or Kv7.5/3) (C) channel currents. (D) Retigabine induced recovery (Ir) of Kv7 (M) current at −20 mV after XE991 application (Ix) expressed as a percentage of the control (vehicle) current (Iv). Recovery calculated as (Ir-Ix)/(Iv-Ix). Data are presented as mean ± S.E.M.; ***P < 0.001 (independent measures two-tailed t-test).

Fig. 5

AVP-induced Ca²⁺ oscillations can be abolished by Kv7 activator, L- and T-type Ca²⁺ channel blockers. (A) Representative example trace showing Ca²⁺ oscillations (indicated as F340/F380 ratio units; r.u.) evoked by the physiological concentration of vasoactive hormone, vasopressin (AVP; 100 pM) in A7r5 cells. (B-D) Example traces of AVP-induced Ca²⁺ transients recorded in the presence of Kv7 channel opener, retigabine (10 μM; B), L-type (nifedipine; 2 μM; C) or T-type (NNC 55-0396; 3 μM; D) Ca²⁺ channel blockers (as indicated). (E) Upper panel: superimposed are Fura2 ratiometric Ca²⁺ recording (black) and FuoVolt membrane potential recording (measured as ΔF/F₀; red) during application of AVP (100 pM) and retigabine (10 μM) during periods indicated by horizontal bars. Lower panel: Cross correlation of the normalised ratiometric Ca²⁺ signal with the normalised FluoVolt signal indicated the time lag between the signals at which the peak correlation occurred. (F-H) Bar graphs summarising the effects of retigabine (F), nifedipine (G) or NNC 55-0396 (H) on the AVP-induced Ca²⁺ spike frequency (spikes/s). Control is the spike frequency in the presence of AVP measured from the onset of the first spike. For quantification of drug effect spike frequency was calculated from the onset of the drug application and until the end of the application of AVP. In panels F-H data are presented as mean ± S.E.M.; n.s., not significant; **P < 0.01 (n = 5).

Fig. 6

AVP-induced Ca²⁺ oscillations are reduced by inhibition of PLC and ER Ca²⁺ release channels. (A) Representative example trace showing Ca²⁺ oscillations (indicated as F340/F380 ratio units; r.u.) evoked by AVP (100 pM) in A7r5 cells pretreated with the phospholipase C (PLC) inhibitor, edelfosine (10 μM). (B-D) Bar graphs summarising the effects of edelfosine on the peak Ca²⁺ level (B), Ca²⁺ response amplitude (ΔR; C) and Ca²⁺ spike frequency (spikes/s) (D) induced by AVP. (E) Example traces of AVP-induced Ca²⁺ transients recorded in the presence of 2-APB (IP₃Rs inhibitor; 100 μM) or tetracaine (RyRs inhibitor; 100 μM), as indicated. (F,G) Bar graphs summarising the effects of the effects of 2-APB or tetracaine on the peak Ca²⁺ level (F) and Ca²⁺ response amplitude (ΔR; G) induced by AVP. In panels B-D and F-G data are presented as mean ± S.E.M.; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (panel B-D, Control, n = 7, edelfosine, n = 10; panel F-G, n = 5).

Fig. 7

Ca²⁺ oscillations induced by Kv7 channel inhibition are insensitive to PLC inhibition. (A) Representative example trace showing Ca²⁺ oscillations (indicated as F340/F380 ratio units; r.u.) evoked by XE991 (10 μM) in A7r5 cells pretreated with PLC inhibitor, edelfosine (10 μM). (B-D) Bar graphs summarising the effects of edelfosine on the peak Ca²⁺ level (B), Ca²⁺ response amplitude (ΔR; C) and Ca²⁺ spike frequency (spikes/s) (D) induced by XE991. In panels B-D data are presented as mean ± S.E.M.; n.s., not significant; (n = 5).

Fig. 8

AVP-induced Ca²⁺ transients in human IMA SMCs is reduced by Kv7 activator, L- and T-type Ca²⁺ channel blockers. (A) Representative example traces showing rises in [Ca²⁺]_i (indicated as F340/F380 ratio units; r.u.) evoked by AVP (100 pM; the application period is indicated by the solid bar) in control conditions (black) or in the presence of Kv7 channel opener, retigabine (10 μM; green), L-type (nifedipine; 2 μM; blue) or T-type (NNC 55-0396; 3 μM; red) Ca²⁺ channel blockers (as indicated). (B,C) Bar graphs showing the peak Ca²⁺ level (B) and mean area under the curve of the response (C) to AVP. (D) Agarose gels stained with SYBR safe to visualise the RT-PCR products corresponding to L-type (CACNA1C, CACNA1D) and T-type (CACNA1 G, CACNA1H, CACNA1I) VGCCs genes. (E) Quantification of RT-PCR results exemplified in panel D, expression is normalised to that of a housekeeping gene, HPRT1 (hypoxanthine phosphoribosyltransferase 1). In panels B, C and E data are presented as mean ± S.E.M.; **P < 0.01, ***P < 0.001 (panels B,C, n≥4; panel E, n≥3).

Fig. 9

Functional expression of Kv7 channels in primary human IMA cells. (A) Representative example trace showing rises in [Ca²⁺]_i (indicated as F340/F380 ratio units; r.u.) evoked by XE991 (10 μM; the application period is indicated by the solid bar). (B) Comparison of Ca²⁺ signals (area under the curve) induced by AVP and XE991 in IMA cells. (C) Agarose gels stained with SYBR safe to visualise the RT-PCR products of KCNQ1, KCNQ2, KCNQ3, KCNQ4 and KCNQ5. (D) Quantification of RT-PCR results exemplified in panel C, expression is normalised to that of a housekeeping gene, HPRT1 (hypoxanthine phosphoribosyltransferase 1). In panels B and D data are presented as mean ± S.E.M.; ***P < 0.001, ****P < 0.0001 (n≥4).