Vascular Kv7 channels control intracellular Ca2+ dynamics in smooth muscle

Figures

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  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).

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  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…

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  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).

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  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).

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  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…

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  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).

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  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).

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  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).

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  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).