Calmodulin kinase and a calmodulin-binding 'IQ' domain facilitate L-type Ca2+ current in rabbit ventricular myocytes by a common mechanism.

1. Ca2+-calmodulin-dependent protein kinase II (CaMK) and a calmodulin (CaM)-binding 'IQ' domain (IQ) are both implicated in Ca2+-dependent regulation of L-type Ca2+ current (I(Ca)). We used an IQ-mimetic peptide (IQmp), under conditions in which CaMK activity was controlled, to test the relationship between these CaM-activated signalling elements in the regulation of L-type Ca2+ channels (LTCCs) and I(Ca) in rabbit ventricular myocytes. 2. A specific CaMK inhibitory peptide nearly abolished I(Ca) facilitation, but the facilitation was 'rescued' by cell dialysis with IQmp. 3. IQmp significantly enhanced I(Ca) facilitation and slowed the fast component of I(Ca) inactivation, compared with an inactive control peptide. Neither effect could be elicited by a more avid CaM-binding peptide, suggesting that generalized CaM buffering did not account for the effects of IQmp. 4. I(Ca) facilitation was abolished and the fast component of inactivation eliminated by ryanodine, caffeine or thapsigargin, suggesting that the sarcoplasmic reticulum (SR) is an important source of Ca2+ for I(Ca) facilitation and inactivation. IQmp did not restore I(Ca) facilitation under these conditions. 5. Engineered Ca2+-independent CaMK and IQmp each markedly increased LTCC open probability (P(o)) in excised cell membrane patches. The LTCC P(o) increases with CaMK and IQmp were non-additive, suggesting that CaMK and IQmp are components of a shared signalling pathway. 6. Both CaMK and IQmp induced a modal gating shift in LTCCs that favoured prolonged openings, indicating that CaMK and IQmp affect LTCCs through a common biophysical mechanism. 7. These findings support the hypothesis that CaMK is required for physiological I(Ca) facilitation in cardiac myocytes. Both CaMK and IQmp were able to induce a modal gating shift in LTCCs, suggesting that each of these signalling elements is important for Ca2+-CaM-dependent LTCC facilitation in cardiac myocytes.

Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels.

A dynamic positive feedback mechanism, known as 'facilitation', augments L-type calcium-ion currents (ICa) in response to increased intracellular Ca2+ concentrations. The Ca2+-binding protein calmodulin (CaM) has been implicated in facilitation, but the single-channel signature and the signalling events underlying Ca2+/CaM-dependent facilitation are unknown. Here we show that the Ca2+/CaM-dependent protein kinase II (CaMK) is necessary and possibly sufficient for ICa facilitation. CaMK induces a channel-gating mode that is characterized by frequent, long openings of L-type Ca2+ channels. We conclude that CaMK-mediated phosphorylation is an essential signalling event in triggering Ca2+/CaM-dependent ICa facilitation.

Ultrafast inactivation causes inward rectification in a voltage-gated K(+) channel from Caenorhabditis elegans.

The exp-2 gene in the nematode Caenorhabditis elegans influences the shape and duration of the action potential of pharyngeal muscle cells. Several loss-of-function mutations in exp-2 lead to broadening of the action potential and to a concomitant slowing of the pumping action of the pharynx. In contrast, a gain-of-function mutation leads to narrow action potentials and shallow pumping. We cloned and functionally characterized the exp-2 gene. The exp-2 gene is homologous to genes of the family of voltage-gated K(+) channels (Kv type). The Xenopus oocyte-expressed EXP-2 channel, although structurally closely related to Kv-type channels, is functionally distinct and very similar to the human ether-à-gogo-related gene (HERG) K(+) channel. In response to depolarization, EXP-2 activates slowly and inactivates very rapidly. On repolarization, recovery from inactivation is also rapid and strongly voltage-dependent. These kinetic properties make the Kv-type EXP-2 channel an inward rectifier that resembles the structurally unrelated HERG channel. Apart from many similarities to HERG, however, the molecular mechanism of fast inactivation appears to be different. Moreover, the single-channel conductance is 5- to 10-fold larger than that of HERG and most Kv-type K(+) channels. It appears that the inward rectification mechanism by rapid inactivation has evolved independently in two distinct classes of structurally unrelated, voltage-gated K(+) channels.

Expression of Ca2+ channels from rat brain with model phenylketonuria in Xenopus oocytes.

Ca2+ channels expressed in Xenopus oocytes using mRNA purified from the brain of the rats subjected to chronic treatment with l-phenylalanine in order to model conditions typical for the congenital disease called phenylketonuria (PKU) were studied using double microelectrode technique. The amplitude of Ca2+ channel currents (IBa, 40 mM Ba2+ as a charge carrier) directed in the oocytes by mRNA from the brain of the animals with model PKU was significantly smaller compared to the control animals (145+/-23 nA vs. 270+/-38 nA, p<0.025) while the voltage-dependence of both currents was similar and typical for that of high voltage-activated (HVA) Ca2+ channels. No evidence for the expression of low voltage-activated Ca2+ channels were found. The decrease of the overall HVA Ba2+ current under model PKU occurred primarily at the expense of the decaying, omega-conotoxin-sensitive component which accounted for about 64% of the total current amplitude in control, and apparently was associated with the activity of the expressed N-type Ca2+ channels. omega-Aga-IVA-sensitive, P/Q component of IBa that contributed not more than 10% to the total current in control showed no change under PKU conditions. In addition to the decreased amplitude, Ba2+ current from model PKU animals showed accelerated run-down during prolonged recording (50%/h compared to 15%/h in control). Our data suggest that hyperphenylalaninemic conditions affect the expression of preferentially N-type Ca2+ channels via the reduction of their specific mRNA content as well as influence the type and manner of channels regulation. The underexpression of N-type Ca2+ channels is consistent with the decrease in the overall number of synaptic contacts during PKU and may be one of the factors contributing to the severe damage of the brain function.

Characterization of hypothalamic low-voltage-activated Ca channels based on their functional expression in Xenopus oocytes.

Ca-channel currents expressed in Xenopus oocytes by means of messenger RNA extracted from rat thalamohypothalamic complex were studied using the double microelectrode technique. Currents were recorded in Cl(-)-free extracellular solutions with 40 mM Ba2+ as a charge carrier. In response to depolarizations from a very negative holding potential (Vh = -120 mV), inward Ba2+ current activated at around -80 mV, peaked at -30 to -20 mV and reversed at +50 mV indicating that it may be transferred through the low voltage-activated calcium channels. The time-dependent inactivation of the current during prolonged depolarization to -20 mV was quite slow and followed a single exponential decay with a time-constant of 1550 ms and a maintained component constituting 30% of the maximal amplitude. The current could not be completely inactivated at any holding potential. As expected for low voltage-activated current, steady-state inactivation curve shifted towards negative potentials. It could be described by the Boltzmann equation with half inactivation potential -78 mV, slope factor 15 mV and maintained level 0.3. Expressed Ba2+ current could be blocked by flunarizine with Kd = 0.42 microM, nifedipine, Kd = 10 microM, and amiloride at 500 microM concentration. Among inorganic Ca-channel blockers the most potent was La3+ (Kd = 0.48 microM) while Cd2+ and Ni2+ were not very discriminative and almost 1000-fold less effective than La3+ (Kd = 0.52 mM and Kd = 0.62 mM, respectively). Our data show that messenger RNA purified from thalamohypothalamic complex induces expression in the oocytes of almost exclusively low voltage-activated calcium channels with voltage-dependent and pharmacological properties very similar to those observed for T-type calcium current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.

Expression of low-voltage activated Ca2+ channels from rat brain neurones in Xenopus oocytes.

Xenopus laevis oocytes were injected with total mRNA obtained from the thalamo-hypothalamic complex of adult rats. In 19 out of 32 injected oocytes a Ba2+ current was expressed after 4 days which could be activated at depolarizations to -70 mV from a holding potential of -120 mV and reached a maximum value at between -30 and -20 mV. The current inactivated monoexponentially with a time constant of about 420 +/- 10 ms (n = 6); its steady state inactivation had a half value of -78 +/- 1 mV (n = 8) and a slope (k) of 11.5 +/- 3.0. These characteristics are typical of LVA (T-type) Ca2+ channels in neurones from the corresponding brain structures, except for the much slower time course of inactivation. These currents were blocked by pharmacological antagonists specific for LVA channels (amiloride, flunarizine), but remained resistant to omega-Aga-IVA and omega-Cg-toxin. These results show that LVA Ca2+ channels can be expressed in oocytes provided that the corresponding mRNA is taken from brain neurones in which they are naturally well expressed.