Calcium sparklets regulate local and global calcium in murine arterial smooth muscle

Abstract

In arterial smooth muscle, protein kinase Calpha (PKCalpha) coerces discrete clusters of L-type Ca2+ channels to operate in a high open probability mode, resulting in subcellular domains of nearly continual Ca2+ influx called 'persistent Ca2+ sparklets'. Our previous work suggested that steady-state Ca2+ entry into arterial myocytes, and thus global [Ca2+]i, is regulated by Ca2+ influx through clusters of L-type Ca2+ channels operating in this persistently active mode in addition to openings of solitary channels functioning in a low-activity mode. Here, we provide the first direct evidence supporting this 'Ca2+ sparklet' model of Ca2+ influx at a physiological membrane potential and external Ca2+ concentration. In support of this model, we found that persistent Ca2+ sparklets produced local and global elevations in [Ca2+]i. Membrane depolarization increased Ca2+ influx via low-activity and high-activity persistent Ca2+ sparklets. Our data indicate that Ca2+ entering arterial smooth muscle through persistent Ca2+ sparklets accounts for approximately 50% of the total dihydropyridine-sensitive (i.e. L-type Ca2+ channel) Ca2+ influx at a physiologically relevant membrane potential (-40 mV) and external Ca2+ concentration (2 mm). Consistent with this, inhibition of basal PKCalpha-dependent persistent Ca2+ sparklets decreased [Ca2+]i by about 50% in isolated arterial myocytes and intact pressurized arteries. Taken together, these data support the conclusion that in arterial smooth muscle steady-state Ca2+ entry and global [Ca2+]i are regulated by low-activity and PKCalpha-dependent high-activity persistent Ca(2+) sparklets.

Figure 1. Simultaneous recordings of Ca²⁺ sparklets and the underlying I_Ca in arterial myocytes

A, Ca²⁺ current (I_Ca), change in total fluorescence intensity (ΔF_total), and ΔF/F₀ records from two representative Ca²⁺ sparklet sites recorded at −70 mV (a and b, centre traces). The lower and upper traces show the integral of ΔF_total and I_Ca, respectively. B, plot of the relationship between Ca²⁺ sparklets signal mass (i.e. peak ΔF_total dt) and ΔQ_Ca in arterial myocytes. The continuous line is the best least squares fit to the data using a linear equation y=mx+b, where m is the slope (555 peak ∫ΔF_totaldt units fC⁻¹), and b is the y intercept.

Figure 2. Ca²⁺ influx is greater via persistent high nP_s Ca²⁺ sparklets than via low nP_s Ca²⁺ sparklets, and increases with membrane depolarization

A, ΔF_max traces from representative low and high nP_s (where n is the number of quantal levels and P_s is the probability that a quantal Ca²⁺ sparklet event is active) Ca²⁺ sparklet sites at −70 (top) and −40 mV (bottom). Amplitude histograms of ΔQ_Ca from low and high nP_s Ca²⁺ sparklet sites at −70 (B) and −40 mV (C). D, scatter plot of ΔQ_Ca amplitudes from low and high nP_s Ca²⁺ sparklet sites at −70 and −40 mV; the red line represents the median value for each column of values.

Figure 3. The duration of high nP_s Ca²⁺ sparklet events is longer than low nP_s Ca²⁺ sparklet events, and increases with membrane depolarization

Histograms of the duration of low (top) and high (bottom) nP_s Ca²⁺ sparklet events at −70 (A) and −40 mV (B). The continuous black line in the low nP_s histograms is a fit to the data with the single exponential function y=Ae^(−x/τ)+y_i, where A is the amplitude (at −70 mV = 0.60; at −40 mV = 0.35), τ is the time constant (at −70 mV = 26 ms; at −40 mV = 41 ms), and y_i is the y intercept (at −70 mV = 0.009; at −40 mV = 0.009). The continuous black line in the high nP_s histograms is a fit to the data with the double exponential function y=A1e^(−x/τ₁)+A2e^(−x/τ₂)+y_i, where A1 is the amplitude of the first component (at −70 mV = 0.30; at −40 mV = 0.17), A2 is the amplitude of the second component (at −70 mV = 0.05; at −40 mV = 0.003), τ₁ is the fast time constant (at −70 mV = 30 ms; at −40 mV = 35 ms), τ₂ is the slow time constant (at −70 mV = 75 ms; at −40 mV = 200 ms), and y_i is the y intercept (at −70 mV = 0.001; at −40 mV = 0.001).

Figure 4. Ca²⁺ sparklets modulate local and global [Ca²⁺]_i

[Ca²⁺]_i records from a control cell (A), a nifedipine (10 μm)-treated cell (B), and a cell dialysed with 100 μm PKCi (C), while in a nominally Ca²⁺-free solution and after the introduction of 2 mm Ca²⁺ external Ca²⁺. The red traces represent the time course of the spatially averaged, global [Ca²⁺]_i; the black trace in the control cell represents the time course of [Ca²⁺]_i at a Ca²⁺ sparklet site (i.e. local [Ca²⁺]_i). D, plot of the mean ±s.e.m. steady-state global [Ca²⁺]_i in control, nifedipine-treated, and PKCi-dialysed myocytes.

Figure 5. Basal PKCα activity increases I_Ca and [Ca²⁺]_i in arterial myocytes

A, representative I_Ca traces (evoked by a voltage pulse from −70 to +30 mV) before (top) and after application of the PKC inhibitor Gö6976 (200 nm; bottom). B, plot of the mean ±s.e.m. peak I_Ca before and after application of Gö6976. C, representative DHP-sensitive traces (I_Ca) evoked during a voltage ramp from −70 to +40 mV with a rate of depolarization of 40 mV s⁻¹ before (top) and after application of Gö6976 (bottom). The continuous line marks the zero current level. The bar graph depicts the mean ±s.e.m. of the total charge (pC) associated with the DHP-sensitive I_Ca recorded under control conditions and after Gö6976. D, global [Ca²⁺]_i records from a typical myocyte (holding potential −40 mV) before and after the application of nifedipine in the absence (i.e. control; top) and presence of Gö6976 (bottom). The bar graph plots the mean ±s.e.m. of the DHP-sensitive component of global Ca²⁺ in control and Gö6976-treated cells.

Figure 6. PKCα modulates arterial wall [Ca²⁺]_i in intact pressurized arteries

A, arterial wall [Ca²⁺]_i in pressurized (80 mmHg) WT and PKCα^−/− mesenteric arteries during application of Gö6976 (200 nm) and isradipine (1 μm). The I_Ca voltage-clamp records shown on the right were obtained from wild-type and PKCα^−/− myocytes during step a depolarization from −70 to +30 mV. B, plot of the mean ±s.e.m. of [Ca²⁺]_i under control conditions (baseline) and after Gö6976, isradipine, and Gö6976 + isradipine in wild-type and PKCα^−/− arteries. C, plot of the mean ±s.e.m. of arterial wall [Ca²⁺]_i under control conditions (baseline) and after application of Gö6976, isradipine, and Gö6976 + isradipine in WT and PKCα^−/− arteries.