Enhanced activity of multiple TRIC-B channels: an endoplasmic reticulum/sarcoplasmic reticulum mechanism to boost counterion currents.

KEY POINTS: There are two subtypes of trimeric intracellular cation (TRIC) channels but their distinct single-channel properties and physiological regulation have not been characterized. We examined the differences in function between native skeletal muscle sarcoplasmic reticulum (SR) K+ -channels from wild-type (WT) mice (where TRIC-A is the principal subtype) and from Tric-a knockout (KO) mice that only express TRIC-B. We find that lone SR K+ -channels from Tric-a KO mice have a lower open probability and gate more frequently in subconducting states than channels from WT mice but, unlike channels from WT mice, multiple channels gate with high open probability with a more than six-fold increase in activity when four channels are present in the bilayer. No evidence was found for a direct gating interaction between ryanodine receptor and SR K+ -channels in Tric-a KO SR, suggesting that TRIC-B-TRIC-B interactions are highly specific and may be important for meeting counterion requirements during excitation-contraction coupling in tissues where TRIC-A is sparse or absent. ABSTRACT: The trimeric intracellular cation channels, TRIC-A and TRIC-B, represent two subtypes of sarcoplasmic reticulum (SR) K+ -channel but their individual functional roles are unknown. We therefore compared the biophysical properties of SR K+ -channels derived from the skeletal muscle of wild-type (WT) or Tric-a knockout (KO) mice. Because TRIC-A is the major TRIC-subtype in skeletal muscle, WT SR will predominantly contain TRIC-A channels, whereas Tric-a KO SR will only contain TRIC-B channels. When lone SR K+ -channels were incorporated into bilayers, the open probability (Po) of channels from Tric-a KO mice was markedly lower than that of channels from WT mice; gating was characterized by shorter opening bursts and more frequent brief subconductance openings. However, unlike channels from WT mice, the Po of SR K+ -channels from Tric-a KO mice increased as increasing channel numbers were present in the bilayer, driving the channels into long sojourns in the fully open state. When co-incorporated into bilayers, ryanodine receptor channels did not directly affect the gating of SR K+ -channels, nor did the presence or absence of SR K+ -channels influence ryanodine receptor activity. We suggest that because of high expression levels in striated muscle, TRIC-A produces most of the counterion flux required during excitation-contraction coupling. TRIC-B, in contrast, is sparsely expressed in most cells and, although lone TRIC-B channels exhibit low Po, the high Po levels reached by multiple TRIC-B channels may provide a compensatory mechanism to rapidly restore K+ gradients and charge differences across the SR of tissues containing few TRIC-A channels.

Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC-A.

KEY POINTS: The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric-a gene knockout (KO) alters the single-channel function of skeletal RyR (RyR1). We find that RyR1 from Tric-a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+ , ATP, caffeine and luminal Ca2+ . In the presence of Mg2+ , ATP cannot effectively activate RyR1 from Tric-a KO mice. Additionally, RyR1 from Tric-a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of ß-adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+ -release. The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+ -release observed in skeletal muscle from Tric-a KO mice, further highlighting the importance of TRIC-A for normal physiological regulation of SR Ca2+ -release in skeletal muscle. ABSTRACT: The type A trimeric intracellular cation channel (TRIC-A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric-a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric-a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage-clamp conditions. We find that RyR1 channels from Tric-a KO mice respond normally to cytosolic Ca2+ , ATP, adenine, caffeine and to luminal Ca2+ . However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+ , ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC-A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC-A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC-A.

Mice lacking the intracellular cation channel TRIC-B have compromised collagen production and impaired bone mineralization.

The trimeric intracellular cation (TRIC) channels TRIC-A and TRIC-B localize predominantly to the endoplasmic reticulum (ER) and likely support Ca(2+) release from intracellular stores by mediating cationic flux to maintain electrical neutrality. Deletion and point mutations in TRIC-B occur in families with autosomal recessive osteogenesis imperfecta. Tric-b knockout mice develop neonatal respiratory failure and exhibit poor bone ossification. We investigated the cellular defect causing the bone phenotype. Bone histology indicated collagen matrix deposition was reduced in Tric-b knockout mice. Osteoblasts, the bone-depositing cells, from Tric-b knockout mice exhibited reduced Ca(2+) release from ER and increased ER Ca(2+) content, which was associated with ER swelling. These cells also had impaired collagen release without a decrease in collagen-encoding transcripts, consistent with a defect in trafficking of collagen through ER. In contrast, osteoclasts, the bone-degrading cells, from Tric-b knockout mice were similar to those from wild-type mice. Thus, TRIC-B function is essential to support the production and release of large amounts of collagen by osteoblasts, which is necessary for bone mineralization.

Subconductance gating and voltage sensitivity of sarcoplasmic reticulum K(+) channels: a modeling approach.

Sarcoplasmic reticulum (SR) K(+) channels are voltage-regulated channels that are thought to be actively gating when the membrane potential across the SR is close to zero as is expected physiologically. A characteristic of SR K(+) channels is that they gate to subconductance open states but the relevance of the subconductance events and their contribution to the overall current flowing through the channels at physiological membrane potentials is not known. We have investigated the relationship between subconductance and full conductance openings and developed kinetic models to describe the voltage sensitivity of channel gating. Because there may be two subtypes of SR K(+) channels (TRIC-A and TRIC-B) present in most tissues, to conduct our study on a homogeneous population of SR K(+) channels, we incorporated SR vesicles derived from Tric-a knockout mice into artificial membranes to examine the remaining SR K(+) channel (TRIC-B) function. The channels displayed very low open probability (Po) at negative potentials (≤0 mV) and opened predominantly to subconductance open states. Positive holding potentials primarily increased the frequency of subconductance state openings and thereby increased the number of subsequent transitions into the full open state, although a slowing of transitions back to the sublevels was also important. We investigated whether the subconductance gating could arise as an artifact of incomplete resolution of rapid transitions between full open and closed states; however, we were not able to produce a model that could fit the data as well as one that included multiple distinct current amplitudes. Our results suggest that the apparent subconductance openings will provide most of the K(+) flux when the SR membrane potential is close to zero. The relative contribution played by openings to the full open state would increase if negative charge developed within the SR thus increasing the capacity of the channel to compensate for ionic imbalances.

Facilitated hyperpolarization signaling in vascular smooth muscle-overexpressing TRIC-A channels.

The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca(2+) release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca(2+) release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca(2+) transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca(2+) sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca(2+) sparks were facilitated and cell surface Ca(2+)-dependent K(+) channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca(2+) channels were inactivated, and thus, the resting intracellular Ca(2+) levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca(2+) signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.