HCN channels: Hyperpolarization-activated cation currents

Hyperpolarization-activated cation currents, termed If, Ih, or Iq, were initially discovered in heart and nerve cells over 20 years ago. These currents contribute to a wide range of physiological functions, including cardiac and neuronal pacemaker activity, the setting of resting potentials, input conductance and length constants, and dendritic integration. The hyperpolarization-activated, cation nonselective (HCN) gene family encodes the channels that underlie Ih.

Unlike most voltage-gated channels, Ih channels are activated by hyperpolarizing voltage steps to potentials negative to −60 mV, near the resting potentials of most cells. This property earned them the designation of If for “funny” or IQ for “queer”

Ih is perhaps most widely known for its proposed role in the generation of spontaneous pacemaker activity, both in the heart and central nervous system. As a result, Ih is often referred to as the pacemaker current. However, it is clear that the spontaneous firing of certain cells, such as Purkinje neurons in the cerebellum and respiratory neurons in the brainstem , do not require the participation of Ih to generate automaticity. Even in the heart, the importance of Ih as the prime generator of depolarizing pacemaker current has been questioned.

Noma & Irisawa first reported the existence in sino-atrial node (SAN) tissue of a slow, time-dependent inward current that was activated by membrane hyperpolarization, later termed Ih. DiFrancesco and colleagues first provided a detailed characterization of this current, which they named If. Shortly thereafter, a similar hyperpolarization-activated cation current was identified in rod photoreceptors and hippocampal pyramidal neurons, which was termed IQ.

Ih is a mixed cation current that typically activates with hyperpolarizing steps to potentials negative to −50 to −60 mV. The kinetics of activation during a hyperpolarization, and deactivation following repolarization, are complex. Activation is usually preceded by a significant delay, resulting in a marked sigmoidal time course of onset for Ih. Following this delay, channel opening can be empirically described by either a single or double exponential function, depending on the cell type. These exponential kinetics also vary widely among different cells. In heart and thalamic relay neurons, the time course of activation is quite slow, requiring several seconds to reach a steady state. In hippocampal CA1 neurons by contrast, the kinetics of activation are quite rapid, with time constants of activation on the order of 30 to 60 ms.

The Ih channel has unusual ion selectivity in that it conducts both Na+ and K+ ions but excludes Li+. Divalent cations neither permeate nor block the channel. The ratio of the K+ to Na+ permeability of the channel, PK:PNa, ranges from 3:1 to 5:1, yielding values for the reversal potential of −25 to −40 mV. As a result, activation of the channel at typical resting potentials results in a net inward current carried largely by Na+, which will depolarize the membrane toward threshold for firing an action potential. One other unusual feature of the channel is that its conductance is highly sensitive to external K+ levels. Reduction of K+ below normal extracellular levels can result in a dramatic decrease in current magnitude. Ih channels also have a very small single channel conductance. Even in elevated external K+, channel conductance is only 1 pS. The sensitivity of Ih to external K+ might provide an important means for regulating Ih function; for example, external K+ elevation during seizure activity or cardiac ischemia might enhance the magnitude of Ih and thus alter excitability.

The characterization of Ih has often been hampered by its relatively small magnitude, combined with the presence of overlapping ionic currents that activate over a similar range of potentials. These currents include inward rectifier K+ currents, persistent voltage-gated Na+ currents (INaP), hyperpolarization-activated Cl currents, and transient A-type K+ currents. Ih has often been distinguished from these other currents by its sensitivity to relatively low concentrations of external Cs+, which produce substantial (>50%) blockade at a concentration of 1–2 mM, and by its insensitivity to 1–2 mM external Ba2+, a potent blocker of inward rectifier K+ channels. However, Cs+ has the drawback of blocking certain K+ channels at this concentration. A number of organic compounds have been described that block Ih fairly specifically. These include ZD-7288, UL-FS49 (zatebradine), and S-16257 (ivabradine), although zatebradine also blocks K+ channels, and ZD-7288 and DK-AH 269 (which is structurally similar to zatebradine) alter synaptic transmission, independently of blocking Ih.


One of the most interesting and important characteristics of Ih is its regulation by cyclic nucleotides. Neurotransmitters that elevate cAMP levels greatly facilitate the activation of Ih by shifting its voltage dependence of gating to more positive potentials, typically by 10 mV or more. As a result, during a hyperpolarizing step to a given voltage, Ih activates more completely and more rapidly. Conversely, neurotransmitters that downregulate cAMP depress the activation of Ih, shifting its activation curve to more negative potentials. DiFrancesco & Tortora made the surprising discovery that the regulation of Ih by cAMP does not require protein phosphorylation. Rather, because cAMP enhanced channel opening in cell-free membrane patches in the absence of MgATP, DiFrancesco & Tortora concluded that Ih gating is directly regulated by cAMP binding.

The regulation of Ih through cyclic nucleotides contributes to the speeding of the heart rate in response to β-adrenergic agonists (which raise levels of cAMP) and to the slowing of the heart rate in response to muscarinic acetylcholine receptor agonists (which reduce levels of cAMP). In the brain, a number of transmitters have been shown to regulate Ih in different neurons through either enhancing or diminishing cAMP levels. Ih can also be regulated in both brain and heart by nitric oxide, which stimulates soluble guanylate cyclase and elevates cGMP levels. Subsequent studies have suggested that the activity of Ih may also be regulated by protein phosphorylation and dephosphorylation. A number of protein kinases (PKs) have been implicated, including PKA, PKC and tyrosine kinases. However, direct evidence for Ih channel phosphorylation is so far lacking.

PHYSIOLOGICAL ROLE OF Ih: GENERAL PRINCIPLES
At least four physiological roles have been ascribed to Ih: (a) control of pacemaker activity (in both heart and brain), (b) control and limitation of resting potential, (c) control of membrane resistance and dendritic integration, and (d) regulation of synaptic transmission.

In a cell at rest, tonic activation of Ih helps set the level of resting potential at a somewhat depolarized level. In addition, activation of Ih also contributes to the resting membrane conductance. Thus the presence of Ih tends to decrease a cell's input resistance, membrane time constant, and length constant.

MOLECULAR BASIS FOR Ih IN BRAIN

All four HCN isoforms are expressed in the mammalian brain. Of these four, HCN3 shows the weakest expression. HCN2 shows a broad pattern of strong mRNA expression and is present in most brain regions. HCN1 is more selectively expressed. For example, it is prominent in layer 5 pyramidal neurons of the neocortex, but not in other cortical layers. This mRNA expression pattern is consistent with the high density of Ih current recorded in the layer 5 neurons.

HCN expression patterns have been particularly well characterized in the hippocampus. There, HCN1 is found in CA1 and CA3 pyramidal neurons, with somewhat higher levels of expression in CA1 neurons compared with that in CA3 neurons. HCN2, in contrast, is expressed at somewhat higher levels in CA3 than in CA1 pyramidal neurons. HCN1 and HCN2 are also expressed in scattered neurons in the stratum oriens and stratum lucidum regions of the hippocampus. Such cells most likely represent inhibitory interneurons, which have been shown to contain Ih. Expression of HCN1 in the dentate gyrus is quite low, especially in the mouse brain. HCN4 is only weakly expressed in hippocampus and neocortex.

HCN1 is also strongly expressed in the inhibitory basket cells and Purkinje neurons of the cerebellum. In the thalamus, high levels of expression of HCN2 and HCN4 are found in the excitatory thalamocortical relay neurons, whereas only HCN2 is found in the inhibitory thalamic reticular neurons.

MOLECULAR BASIS FOR Ih IN HEART

HCN1, HCN2, and HCN4 are expressed in heart. Their relative mRNA abundance varies with cardiac region, species, age, and, perhaps, disease state. Although there is good correlation between absolute level of HCN message and magnitude of measured Ih among cardiac regions and species, it has proven difficult to explain functional heterogeneity in the heart with specific isoform expression patterns. In this regard, heart differs from brain in that cardiac Ih exhibits a much wider range of regional voltage dependence.

The SAN, the normal pacemaking region of the heart, exhibits both the largest and most positively activating pacemaker current and expresses the highest message level. HCN4 is the most predominant isoform, accounting for >80% of the total HCN mRNA. Significant levels of HCN1 are present in rabbit SAN (20% of total HCN mRNA) but only very low levels are detected in mouse. In dog SAN, low levels of HCN1 (7%) and HCN2 (8%) mRNA make up the remainder of HCN transcripts.

HCN4 is also the predominant HCN transcript in cardiac Purkinje fibers, specialized conducting tissue that can exhibit subsidiary pacemaker activity. Canine Purkinje fibers, which exhibit significant automaticity and display a large Ih, show the highest level of HCN mRNA expression in the heart outside of the SAN (35% of SAN). HCN4 accounts for 90% of the transcripts, with the remainder contributed by HCN2. In contrast, rabbit Purkinje fibers, which tend not to be automatic and exhibit little Ih, show minimal levels of HCN message (4% of SAN). The HCN mRNA that is present represents roughly equivalent levels of HCN1 and HCN4, with a minor contribution of HCN2 (10%).

In the rabbit ventricle only HCN2 is detected; however, overall levels are extremely low and no measurable Ih is found. Canine and rat ventricle exhibit greater Ih and higher HCN levels than rabbit. HCN2 is by far the predominant ventricular isoform, especially in adult animals, with the balance being HCN4.

The distinct biophysical properties of the HCN isoforms expressed in different regions of the heart cannot account for the marked differences in the time-dependence and voltage-dependence of activation of native Ih in these same regions. Thus, although Ih in the SAN activates more rapidly than Ih in other cardiac regions, the predominant isoform expressed in SAN, HCN4, is the most slowly activating isoform in heterologous expression systems. Moreover, whereas the V½ values of the four HCN isoforms vary by no more than 20 mV, the V½ values of native Ih currents may vary by as much as 80 mV between SAN and ventricle. During development, the voltage dependence of Ih activation in ventricular muscle shifts by up to 40 mV toward more negative potentials, so that in adults the threshold for Ih activation is negative to the resting potential [although there is some debate as to whether the developmental change is a shift in voltage dependence or a reduction in current magnitude ]. These results suggest that factors within a cardiac cell can influence the voltage dependence of an individual HCN isoform. In fact, when HCN2 is overexpressed in neonatal and adult rat ventricular myocytes, the voltage dependence is more positive in the neonatal cells, and this difference is independent of basal cAMP levels.


Although the biophysical properties of heterologously expressed HCN isoforms cannot fully account for the observed variation in native Ih, there is some correlation between which isoforms are expressed in a specific region and the voltage dependence of the native pacemaker current; regions with the most negative activation (e.g., ventricle) tend to express HCN2 predominantly, whereas regions with more positive voltage ranges of activation express HCN4. In addition, whereas HCN2 is the dominant ventricular isoform throughout development, the relative expression ratio of HCN2:HCN4 increases from 5:1 in the neonatal rat ventricle to 13:1 in the adult rat ventricle at the same time that the voltage dependence of the native Ih is becoming more negative . The large phenotypic variation in Ih throughout the heart may reflect the differential modulation of HCN subunits by factors such as phosphorylation or auxiliary subunits, which may be turned on in distinct regions or at different developmental stages by the action of hormones, transmitters, or growth factors. Thus the rationale for the regional patterns of HCN isoform expression might be a differential susceptibility of these isoforms to such modulatory changes. In this respect, it is interesting that in a β2-adrenergic overexpressing mouse heart, ventricular HCN4 message is upregulated with no change in HCN2.

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