Ca2+ Sparks Act as Potent Regulators of Excitation-Contraction Coupling in Airway Smooth Muscle

Ronghua Zhuge,Rongfeng Bao,Kevin E Fogarty,Lawrence M Lifshitz

doi:10.1074/jbc.m109.067546

Abstract

Ca2+ sparks are short lived and localized Ca2+ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca2+-activated K+ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca2+ channels. But in many smooth muscles from a variety of organs, Ca2+ sparks can additionally activate Ca2+-activated Cl(-) channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca2+ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca2+ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca2+ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca2+ channels, resulting in an increase in global [Ca2+](i) and cell contraction. Therefore, Ca2+ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.

Highlights

We demonstrated that Ca2ϩ sparks under physiological conditions induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations
Ca2ϩ Sparks Induce BiMPTs via Activating BK Channels and ClCa Channels under Physiological Conditions—Ca2ϩ sparks activate only spontaneous transient inward currents (STICs) at EK, only spontaneous transient outward currents (STOCs) at ECl, and spontaneous transient outward and inward currents at potentials between EK and ECl in airway smooth muscle (ASM) from mouse as they do in ASM from other species, and in the smooth muscle from other tissues (7, 14, 15, 17)
In light of the stabilizing effect of Ca2ϩ sparks on the membrane potential, we explored the hypothesis that Ca2ϩ sparks and BiMPTs are the signals that prevent ASM from generating action potential

Summary

EXPERIMENTAL PROCEDURES

Cell Isolation—Male Swiss Webster mice (4 – 6 weeks) were euthanized with intraperitoneal injection of a lethal dose of sodium pentobarbital (50 mg kgϪ1). Note the following: 1) the membrane potential transient is biphasic with a hyperpolarization phase followed by a depolarization phase, so it is designated as BiMPT; 2) the endogenous fixed Ca2ϩ buffer (i.e. 81 ␮M with a Kd of 0.66 ␮M) and following calculations as of estimated in the same type of cells by Bao et al (13) was taken into account in this signal mass and ICa(spark). B, panel i, an original long recording of membrane potential under perforated whole-cell configuration by including 160 ␮g/ml amphotericin B in the patch pipette. Cells were imaged using a custom-built wide field digital imaging system (7), and their lengths were determined by a custom-written software Reagents and Their Application—All chemicals except fluo-3, fura-2 (Invitrogen), and ryanodine (Calbiochem) were purchased from Sigma. Student’s paired t test, and the significance level was set at p Ͻ 0.05

RESULTS

The above assertion is strengthened by the experiments in

DISCUSSION